Hello everyone and welcome to NEMO's webinar, Audience at the Centre, Development Strategies and Organizational Change. My name is Elizabeth and I work for NEMO. As the network for museums in Europe, our main activities include advocating for museums at the EU level, providing training opportunities, providing a platform for museums to exchange and then learn from each other, and helping museums to cooperate across borders. We are looking forward to today's webinar facilitated by Adeste Plus. We will be hearing from Alessandra Garibaldi, head of the Transnational Projects of the Foundationi Fitzcarraldo, Inesh Kamara, founder, CEO and creative and special projects manager at the Mapitas Ideas, and Susanna Gomez da Silva, head of education at the Modern Art Center of the Kaluste Globen-Kian Foundation. The NEMO webinar will present a hands-on methodology for creating, prototyping and testing audience development strategies and implementing organizational change. The methodology, audience-centered experience design, has been developed since 2016 by the Adeste Plus project and strives to reach the real people behind hard data, replacing abstract data points with tangible connections. At the end of the session you will have the opportunity to ask questions using the chat function, or if you'd like you can come on screen and ask your questions live. Please note that the entire session and conversation following will be recorded. So without further ado I'm going to hand this over to our speakers to get started. Thank you Leeds and good morning everybody. I am Alessandra Garibaldi and I will introduce you in the first 10 minutes let's say the frame of the Adeste Plus project and then Inesh Kamara will follow with the going a little bit more in depth with the methodology and Susanna Gomez da Silva will help you see how it actually works in a real museum context. So thank you very much to NEMO for organizing this webinar. This belongs to a series of webinars but we are running across Europe to disseminate and discuss what are our achievements so far towards the end of the project. So I will share with you presentation. So we are 15 partners and the partnership of this project is composed by articulated let's say in different levels which is for us very important because we tried to match research partners artistic partners such as Gulbenken Foundation and many others and policy partners considering that somehow tackling the issue of cultural participation might be different from different perspectives and we did want to somehow enable cultural institutions and museums and theaters and libraries to somehow being able to deliver that side of the story which is audience development so cultural participation is a major challenge much bigger than every cultural institution but we all have a role to play in it and museums have a major role in in this. This is not a specific tool for museums but it has been tested especially in the first in the second round let's say of tests we did not only with artistic partners also with many museums because we wanted this to be a way for all to somehow reflect and find new practices towards audience development. So the project is mainly about you might have seen this video but it's mainly about creating a methodology to try to help cultural institutions and it's quite easy methodology in a way so it's based on design thinking is a series of workshops run within the cultural institutions with the staff a transversal group of staff so to cross cut the different sectors that usually don't work together within institutions and we through this model we tested with them we refined the model and then we tested it again in other countries with other cultural institutions dozens and then we now have what we call a blueprint so basically a process that would be made available for all all the world not just Europe so that other cultural institutions can get inspiration and see how can this could look like. So let's say the principle that I would like to introduce in the very beginning is that in our understanding audience development is not a technical issue not just a technical issue it's a matter of value of principles of way of behaving and especially it's not about developing audiences but mainly about developing cultural organizations meaning that if we failed up to now into the endeavour it's not an undertaking but an endeavour of enlarging cultural participation across Europe in a more diverse deep and engaged relationship with citizens it's also because we are not all equipped not at the moment and although we do many amazing projects in cultural participation particularly museum education in this is a very long story we didn't achieve with and Data told us that kind of participation and this is because there is something we need to change within ourselves as cultural organizations and before we can somehow dream to change something outside for citizens. So we started from the principles that audience development effective let's say audience development meaning a long lasting one that really changes relationships between organizations and citizens but also a lasting one that becomes sustainable are let's say few barriers that we tried to somehow overcome or address through the principles that we use for designing our project the first is that usually audience development is an activity demanded to educators marketers communication managers at some point of the production chain somehow they are sometimes amazing but they are often most of the time let's say tackled as if they were an add-on to the activity is the last mile is never embedded into the design of the programming of displaying of communication and everything so what we did is that we considered that it was not about empowering single participants or single members of the staff in doing amazing things that end up usually to be very aside compared to the let's say full activity and structural activity of organization but to empower change makers throughout the organizations so in the process we basically choose to work with and groups of members of the staff there was one change maker let's say in charge of leading the hours and supporting the say leading the process but all the participants that is a communication manager marketing managers and visitor experience manager programmers curators artistic directors they were all change makers and this because it has to be a shared responsibility and this leads to the second point that it silos practices those amazing projects usually have the problem that they start and end so at the end of the project and at the end of the budget also the relationship with the audience somehow get weaker or disappear or alongside these silos practices are those for which especially in big institutions but also in medium and small site organizations are silos practices so they're my work in the exhibition as exhibition manager and here my work as educator starts here so usually those groups don't work together I mean these sometimes happen but not very often not as often as we would like so the the teams we were working with were as much diverse as possible technicians all you can imagine the third reason for which audience development processes are not successful as hope or expected is that often they are based on stereotypes we are all terribly biased by stereotypes and data sometimes don't help because they are useful absolutely necessary good cementation is needed but it's not enough so going beyond real data to get to real people and getting in touch with that feeling that is the real people needs and diversity is something that can be developed only through empathy and this through the process is what we try to do not just for educators because they are already empathetic even too much but also with technicians with marketing managers with other people within the organization that usually don't have this feeling of for whom and who are the ones they are working for we are almost at the end another issue we see is that we are often as organization let's say self-sustaining self-maintaining we are very risk adverse as institutions so we might have someone more with more risk attitude but as an organizations we are usually very scared of taking risks so the use of the same thinking in this is great also because it helps prototyping and failing fast and failing and going for iterations that makes risk somehow more acceptable and the last is of course the fear of losing control we don't have a receipt for this but the big issue is where is up to what point we are going to allow people in giving their voices hearing their voices into the interpretation also of our collections this is not easy at all and there's no one solution but anyway it's a discussion we must have and throughout the process we tried and also through the other activities of the project we tried to open up this kind of conversation because it's really important to have this conversation otherwise we will be always working on the surface of technicalities somehow if we don't accept and don't discuss and debate within the organization about what's the role of audiences how much power are we going to give up how much space we are actually given to listen without giving up our role our mission as institutions so our challenge was to develop a methodology for transformative audience focus to work considering that we didn't just want to give means to design good experiences for audiences but also through this process to change the way cultural organizations were working internally and perceiving their role coming to the idea of cultural participation so we did what we call a blueprint and the milestones were prototyping the methodology testing it with six cultural organizations across Europe among which there was Calo School Banking then refining tests with other institutions and then organizing summer schools and conferences and this is also the chance for us for inviting you to our final conference I know that some NIMO members also subscribe to the summer school I'm very happy about that and we are very glad and also the conference will be open anyway there will be no selection it will be also online for those who would not be able to attend it in Turing next November and that will be the moment in which we will try really to open up and discuss so what that's a tool just like many European projects are developing around with other means and approaches but this is our contribution so I think that that conference will be really very interesting time to discuss about different approaches to promote cultural participation which are somehow transformative for cultural institutions and so I leave the floor to Ines for the next part of the presentation thank you so hi good morning I'm so happy to be with you here so Alessandro has made the big introduction and as you can see it's a very ambitious project because basically the idea is to open up cultural organization and promote change within by learning with the audiences and the project had this huge journey until this moment that's focused on consolidating the methodology and trying it out to see how it works in different organizational settings and we're talking about sectorial settings so going from theaters to museums but also from the size point of view so we really wanted to to understand if this worked in big organizations but also in smaller organizations and what would be the the challenges about that so the first thing that we have to to know about our framework so to speak about the methodology is that it's based on change and it's based on the three-step model from Lewin and what we want to do is to trust the methodology so that's the first challenge it's to trust the process even when it seems that it's not going to work we also aim for a very transparent a very practical approach hence we are going to launch the blueprint online until the end of the year and it will be like an open source methodology and the first thing is to do is that we know that we have to go for unfreezing we have to move and then we have to re-free so in the process what we're talking about is going from a jump from what is the status quo and our pre-exemptions to a moment that we think together as a collective intelligence and then we go and we try to freeze what we've decided into a prototype that can be that is doable so when we went for the design thinking proud diagram we were looking for that kind of approach and basically we're going to go from moments where we are favoring divergent practices and thoughts to convergent and the process as we call it as a double diamond just goes to open it up have a very fruitful and a very direct discussion about what we're thinking about and then we just close to get conclusions and then we'll open again and then we'll close one of the big important parts about the yeast cycle is that it involves people with different characteristics and profiles and different types of knowledge within the organization and that's one of the main issues of the process so in all these a dash to plus workshops from the artistic partners and research partners to the extended to the waterfall programs we really tried to bring diversity from the organization inside the room and the first step would be to work for in freeze to build up this team this small revolutionary cell within the organization then to try to to work upon empathizing and overcoming what Allison was describing as the stereotypes that we have towards what we do and towards our audiences then define a problem then ideate and put out there all kinds of different ideas and concepts and really go above and then prototype and try to get a feasible project so these are basically the steps from a dish that end with the embedding of the prototype in the organization and that's the magic so to speak of opening up the organization to the audiences and also promoting internal change so here you can see that we go from preparing aligning the team to at the end to do the process of embedding from our experience in all these different workshops and processes it was so interesting because the target audiences that we were usually discussing in the preparation meetings was not the target audience that was defined within this revolutionary cell of the organization so we really felt that there is a difference there is a change when we join people that come from very different departments and also from hierarchical levels in the organizations that bring in this kind of definition and bring in new people so to speak so that leads to what we were talking about this idea of a surprise this idea of really trusting the process and really embracing the all the surprises and all the changes that it can bring in it this it it demands preparatory work of course because you really have to have everybody aligned for the project and that is work you can't just get into a room and just say let's do this now you have to explain the process to the people and you have to build up this team but it's already it's a very creative process and that's also sometimes a challenge for our organizations because people in spite of working in cultural organizations sometimes they feel that they have these barriers to any kind of experimentation taking risks and being creative and that's also part of the preparatory work and this idea of the double diamond scheme where you're always dividing the work between divergent and convergent phases really puts people in the right mood so to speak and then it's all about this idea of prototyping and really what we would wish for would be for any organization cultural or otherwise to be able to develop themselves in these micro or macro prototypes that would be enablers of change and work so next for the next project what we're doing this here is that we're spreading the knowledge spreading our mantra we've really tested the methodology and we know that it really works and our extended waterfall program as alexander described between the summer school the european conferences and the policy forums really have to do with this dissemination of the methodology but also of the project values of what we all share and we really feel that over these terrible last two eighteen months of the pandemics it's really important for us to refocus on the audiences refocus on our relationship with our publics with our visitors to explore the new channels that we opened with the pandemics crisis because we're talking now about this mix of digital audiences with people that come into our doors in the museums and we're also talking about this approach that has to do with serving our mission serving cultural democracy but also having a very strong marketing practical managerial approach and we really think that this will be the right pathway for the projects and we hope to see you again soon you can see more about our work at the a dash plus website and now i'm going to pass the word to susana that's going to talk about the prototype that was developed according to this methodology by the kalusko binkia foundation our artistic partner in Portugal and it was really i'm really happy to be with you today to put susana into the the circle because uh map of the zedayesh and kalusko binkia foundation where the portuguese partners are the portuguese partners of the project and it's been this terrific and very very interesting journey susana i'm just going to pass it on to you hi everyone it's really wonderful to be here and as Ines said we were the partners the portuguese partners of adeste and it was really a great journey so what i would like to start by doing is by presenting a short video that will highlight to the whole process so as you've been following just just before the video as we've been following we created the prototype and i'm i'm here representing not only the kalus foundation but the whole team of change makers so as Alessandro presented we tried to make the teams working within the institution as wider and diverse possible and so uh just please note that this video is the result of a prototype created by different departments inside of the organization working together so the music department the museum and the kalusko binkian museum um and the gardens which has also an audience development plan so please enjoy the video and then i'll get back to you and develop a common house imagine in fact the binkian 1525 imagine is an experimental project basically it's a kind of laboratory that allows us to work with young public thinking about what it is to program for and with young public the image provides the space where this experience can be given we gather a group of young people through open call with which we still have what is programming what are the institutions like the binkian that would come to program and what they frequent while users in the programming institutions and then we give them the opportunity to think with us about what would be programming from their point of view and propose some events that were their word it's a project that brings a group of young people that were invited to come to think in programming especially programming for the faxes and hectares and to program with us the various educational services of the foundation therefore educational service of the music or educational service of the garden and the educational service of the museum and also involving other organic units such as communication and marketing the image here in 25 really proposes to let us do that or listen to us to understand what our concerns were what were our priorities in terms of not only programming but also of current conversations and really give us space to work on this and this is historically a music it's a barbershop that gives us the opportunity to buy these origins and these roots of these cultures and to do the project with what I already read the programming program it's also a part of your program we were in contact with people who introduced Adard, Talks and Converses people from the artistic domain, programmers and it was amazing to have the opportunity to talk to them and to know how it was their journey everything that they showed us and how it works in real life to have this idealized idea of how it is that then broke in some way something that surprised me a lot in a very pleasant way, very positive was the fact of having the perception that these young people come to the programming program a tool to talk about the daily concerns of what is the reality of today's days of what is discussed every day that is, of a very different kind and if we all live in this ecosystem because there are certain stories that never happened then from there it was to think about everything that was going to happen in the world I almost want the systems that we thought to be relevant and we just started to draw the problem we just started to draw the problem I'm going to take a picture now I'm going to take a picture now I'm going to take a picture now I'm going to take a picture now I'm going to take a picture now this set of ideas of programming that they brought in our meeting it was a very, very different set of ideas many ideas were very interesting it was very present the theme of dissident sexualities the colonial issues issues related to racism and to go out and there were already ideas about how to formalize these proposals it would be not only to cross these ideas with what is the ADM of the Foundation how to work with the various educational services really how could they compete in terms of programming then the fact that we met to talk about the connections we have in common all the ideas that people are different because it was interesting people with completely different backgrounds to see what we have in common and there are many things in common when the pandemic came and things got a little stagnant what happened is that many times I realized that the potato we didn't have was only in our hands we, the young people we are lost they will direct us to everything but actually we were all in the same bar when the lockdown came because of the pandemic we had the opportunity to program something for the public and on the digital but as a group we decided to take this time for a break and to deepen a series of reflections and we thought that this was a resistance of course because the opportunity we are living today I think they stopped to think and to deepen themes and reflect it is only a resistance act and we had the opportunity to create a cycle of conversations to talk with a series of artists and researchers related to the themes that we had already been debating and we realized that with the pandemic just becoming more urgent it was an opportunity to give this thought so far we had to continue with the project what happened was a second program which was actually a start it was all connected to digital all virtually but meanwhile we were able to adapt what we already had several things that we had already prepared to this new online situation we developed conversations we reorganized into groups we made the groups, we came back to do it was a lot of discussion a lot of conversation to see them project to sit down with ideas to talk with the artists, to invite and put everything into the process was very interesting and it was really for me the most important moment of the project to this day of the things that I learned and of what I liked the most of this process was the dialogue with other people with other ideas to realize that there are themes that I already know but that I don't know so much about them our group, Imagina is making it very obvious the need for institutions the need for institutions to have to change to have to be more disponible, to have to be more accessible and to have to address issues without being afraid of being critical and this door is really open and it can be hard but it won't close so this door of communication with the world that is out there of the occultation of who is out there and not only the programmers that are inside I think that we all left questions and important questions that should be answered inside the foundation that we really couldn't program we couldn't do anything if we didn't know what was on the other side and this becomes an institution and becomes an accessible program when we have on our table several perspectives of what is happening in the world in a project that puts the public in the center of the equation and not in the sense of the public as a destination but of the public as well as a decision in some way, someone who has voice and who has power to make decisions and to propose institutions have to be able to create flexibility in the sense that the area of negotiation is much bigger when I mean that it is something new it is a sign that maybe the foundation can assume that the risk that the need for risk is a fundamental factor for this change that we so much want the institution that we are in reform, reconstruct recount, reform to create, to give voice and body to be voice and body we want to program the critical brain and here ends the poem so as you probably noticed this was a project that combined several different departments at the Gubengen foundation the Gubengen foundation is a very big organization with many different cultural assets and it has a music department with a whole programming on music and it has also a building and a garden which is classified as heritage and so it is worked also as an audience development plan and then it has two museums two really big collections and so we needed why the team and that's why this project combined people from all these apartments and also the marketing and the communication and our prototype was actually engaging one of the audiences we realized that was the most absent audience in our programming the youngsters which is common to many cultural institutions that I know and so we just decided that this could be our target group to test and to prototype so after all the workshops we've done with our national partners so Mapa does it there we came out with this idea of developing a plan to work in a peer-to-peer way with the younger audiences so one of the goals of these was how could we program for and read the youngsters and how can we engage them in the whole process of deciding things you know making decisions and sharing the communication process and the programming process so it was crucial to listen to debate and to learn with each other and also to create a participatory and collaborative methodology so we just created four major phases so the first one was to listen and that's when we all gathered all these youngsters that came to us by open call so we wanted a very diverse group so we could listen to people that usually wouldn't listen to and listening together and in order to do that inviting not only people from the organization because the change makers although we were all in the process we wanted to contaminate the rest of the organization so we also invited people from the other departments to come in step in and engage with youngsters we also invited people from out there from other cultural institutions just in order to listen the more we could not only to the youngsters but also to the organization we were in but also the cultural field around us and then ideate and think what would happen if I'm now in the role of a programmer what would it be possible what would I like to see in the future and then create the moment to launch and test all the programming and in order to do you know the learning of this then have time to reflect and debate what happened and how does it make us change in the future this is a process of change anyway as you saw the pandemic hit us really hard and it was exactly in the moment where we were beginning to make real everything that was being decided and all the youngsters have decided a whole programming a very ambitious one we had decided that we would go on their ideas and taking all the risks because as you saw programming was seen by the whole group and by this audience as a way of making a stand in the world and just saying what is important to us as a generation as a person what is crucial for the future so the pandemic came in a moment where we were beginning to make it real and so it was really crucial to stop pause reassemble and think what is this about actually the whole process was about change change out there change within us as an organization but also as individuals and so it was very interesting to see that the prototype we created and the methodology we were following with ADESTA plus ACID really created a very flexible approach to programming or to whatever prototype we were trying to create and this open structure was what enabled us to deal with something that we were not expecting and to have the flexibility to adapt and to listen more even more carefully than we were doing and that's how we came up with another way of testing what we wanted to test which was if we design programming all together if these youngsters did the role of the programmer inside this institution what would happen and actually this was possible of course the program was less ambitious than the first one but it was ambitious enough to make us think and reflect on what could happen when we change roles what could happen when change happens it also helped us learning with the process because even with all these changes even with this transformation of a big program with everything in our life and sound and concerts and guest artists and then just make it into a very small version of this with online conversations but even this change didn't stop the reasoning behind the project which was what happened when we worked together with the target audience we wanted to work with when we changed the rules, swept rules but also when we shared the stage when we shared the decision making process and by doing that we learned that probably this will be true for all audiences but for this audience particularly it was a statement or about the young without the young and the reason I say this probably will work for all audiences is what Alessandro said in the beginning we work on stereotypes most of the time and we talk on behalf of people we make decisions on behalf of people and it's quite interesting when we incorporate all these people into the decision making process and when we listen carefully then the other thing we think together and act together so don't use this only as a consultation as a champions group just work together act together give us the stage and share the stage then the listening process it was again another outcome of these because although we were aware that listening was important listening in these levels just in the small group and in the wider group and within the organization outside the organization this made us realize that the more we listen the more we were able to learn and to change then the other one was the participatory processes and methodologies are really crucial if we want to listen as to be more in the center of any institution that is audience centered and focused the key words that youngsters highlighted throughout all the process were openness and dialogue and informality and proximity which is quite interesting for such organizations like Gulbenkin Foundation which are really big organizations and usually perceived as being really serious or very distant in order to incorporate other audiences we also had to change the way we approach even the work atmosphere and the work politics the program is to intervene in society and this was another thing that was said several times throughout the whole process that was actually one of the major demands that these audiences made to us and so we should take more risks we should be aware that what is happening around us what is happening in the world and irrelevant in the now, the here and now thinking about the future and then again some key words that were really important for us identity for this group identity issues were the thing to discuss and we shouldn't avoid difficult uncomfortable themes sustainability critical thinking the generalization of the narratives changing the point of view equality and equity and visibility to the stories that usually are untold so with these I would say that this was testing and prototyping and make real all the the structure of this acid methodology but it was also a way of trying to create a new matrix for the future and I must say that we are currently planning for next year to embed all this learning into a programming that could be regular programming and no longer only prototyping tests for a younger programming in the same model so incorporating youngsters in the decision making process and working with them in order to create a peer-to-peer program that could last particularly for the next three years and then learn with the process. Thank you. I don't know if we are already ready for the questions. Yes, thank you so much, Susana and Alessandra Ines if you both want to come back on screen we'll be happy to open up the Q&A session but firstly thank you all so much for this great presentation and the examples of the program you know this is great to see I wanted to start with a question or two of my own actually Ines you had this diagram up of where it's starting from the preparatory phase to the embedding phase and actually Susana you can probably answer this as well I'm wondering if there are certain points between those where you find that organizations most often have a stumbling block or if it's just completely different for everyone well the shared thing is that everybody does find let's say those steps I mean of course when you see the results that our institution our partners achieved especially when you see a project like Imagina which ambition was really high and actually up to the ambition you might think that everything is easy but it's not actually every step I mean this is a step process step by step process but every step somehow brings you back to the usual issues so all the time in each step you have to renegotiate what is this why we are doing are we letting them in really why they're not listening and others are in so it's a because the main thing the main let's say feature of the design thinking that we fell in love with although I mean it's a tool and it's a design tool everybody's using in many different contexts it's not as such but is that it's a reflective process and it's a relational process so these two levels were absolutely key and this means that all those steps and difficulties happen all along the way I don't know Ines and Susanna if you agree but I think that that's it's not it's an answer but it's not an easy one yeah yeah and I would had that well of course you're just listening to this case but among the artistic partners in Adeste there are so many different situations that they would just make the point for this for this question you know we all stepped in in different ways we all had to go back to the beginning so many times throughout the process but we all ended up with some change and some really good reflection I think the hard parts I must say and still is is the embedded thing you know as we all know you know you test things you you you create a project and you learn from the mistakes you learn from the successes but then make it last and make it really important as a changing tool that's the hard part and I'm really glad that we are going on on this absolutely um it's a different model of working because in a certain sense it's not a normal because you let a foreign organization in in this case Gubingin let Mappa in and all the other partners went through that process so we are the strange element but the goal is not for the strange element to tell you like in the top bottom perspective in the consultancy project what's wrong with you and what you want to do but the goal is that where the strange ingredients is going to shake the waters and put that collective intelligence that's within the organization working so that's the real the challenge and of course as Alessandra and Susanna were saying it's all about acknowledging that there are barriers that things are very difficult but we're in this together and that's a very important thing and if I may answer to the question I see about how to implement the given the blocking forces and and also it is also the story of the connector which was the entry point actually we looked for cultural institutions ready to do that so this was the legacy of an unsuccessful attempt we did the year before 2017 and already then the former director of the museum will banking was quite interested in setting up something like that but the complexity of organizations is quite layered so 300 employees is not 30 employees and it's not five employees and we had more or less in our institutions where but we also tested with a voluntary community art based center in Saragossa for example so you had really the span or the opera theater in Rieke so with many different levels and layers the entry point was us that's true they are blocking but we think that there are plenty of people within cultural organizations really keen to do that maybe they are not in the board of directors sometimes they are in one case we had the director taking parts in all the stages of the process all the stages all the workshop all the employees and the director always there sometimes this happen and sometimes not we are now delivering a model so the prototype is now become a blueprint and it is available on the website it will be available in a dedicated website with the tools the steps and whatever but of course change doesn't happen because we have a tool but if you want the change then you need tools so it's negotiating not everybody was completely aware of that kind of challenge as organization when they stepped in there was someone really keen to and someone else really that was just thinking it's just another audience development project let's try that it's European project let's do but without that understanding of the real implications and of course this also changes in terms of results so our strategy is keep doing this because in 10 years of long lasting cooperation projects working on this kind of things we will push it forward but there are already many many people really many people across Europe professionals really into the topic so we are not just a few ones avant-garde we are many luckily great I also love Inesh what you said earlier about having the foreign element involved I think that is going to require so much trust and vulnerability I think though I mean I would say that we're also within our sector though that we we do have such this communal aspect that we do want to support each other and so it's always great to be able to see that in action I think this is bouncing off a bit of what you were already just discussing Alessandra but there's certainly quite a few questions just on the initial point of the project in terms of you know which channels are used to reach out to potential participants but not so just the technical aspects of implementing this and getting involved could you discuss that a little more do you want for the project or just for the Aleste as a whole I think for your project for me yeah because I looked at there were some really particular questions okay so as you probably noticed getting into the prototype was part of a set of seven capacity workshops we had with MAPPA and that's really important just to mention these because these capacity workshops are the ones that created team and as you notice we were from different departments so one of the most important things to begin with was to align the whole team and just get kind of a common ground although we all work in the same cultural institution we all deal with completely different kinds of programming we set this programming to different kinds of audiences and our priorities sometimes are very different although we think they are the same so just having the opportunity of being together in seven workshops just with this aim of getting together, find common ground and then decide what would be if we could decide on a prototype to try on something that we really want to try and maybe we never did and so this was the beginning then there is another beginning so we get to this and then we have to make these being approved by the board because I notice I know I'm not forgetting what you were asking but there was another question about these blocking forces they could be blocking or they could not be the way then you have these in the structure so by being so many people from different departments actually that made it much more easy to go up and just make the whole institution get along with what we were proposing because suddenly this was not the idea of one department or the museum or the education department but something that we wanted to try out through different people in different perspectives that could cross into different departments and made us better equipped and better prepared to deal with other audiences in the future and this was seen as something really good, very positive point so one of the things is as Ines said and Alessander having the directors involved somehow it's really good because then they are very supportive so one of the things was first prototype was pitched we made a pitch for all the directors involved because there were so many so we made sure that when we made the decision of now we go out there and going to find our youngsters this was already supported by everyone that made decisions inside the institution and took to the board it happened the same in the end so when we had the final result we created this video we had all these findings on the project we took it to the board again we just made a special presentation for all members of the board and all the other directors involved so people were aware of what happened and I must say that this is the reason why we will be able to trade again in the near future so never forget that all the structure needs to feel that this is really important it doesn't matter if it comes from up down or down or down to the bottom then the other part that was asked was how did we find these youngsters and how we decided this so the Gubengen Foundation as I told you is very important it's a very big institution and it's very well known in Portugal but it has no resonance in the youngsters culture to tell the truth the Gubengen Foundation because it's made its way being a kind of a ministry of culture during a lot of time not nowadays but it's really crucial and important relevant for another generation not the younger generation the younger generation is already born in a completely different country with a lot of other cultural institutions with a lot of programming happening everywhere so Gubengen Foundation wouldn't be the first place for these youngsters to come so we were very aware of these and we knew that if we just made a regular open call in the website of the Gubengen Foundation or the Facebook or the social media of the Gubengen we only would get the few youngsters that identified themselves already in this institution and we didn't want to find these ones we wanted to have the other ones the ones who would tell us well I would never go unless you change something otherwise they would come so in order to do this again we went to different departments in the Gubengen Foundation that work in completely different projects that work in the neighborhoods or you know associations with ONG so other kind of people and we asked for help to create a network of contacts that would help us reach people that in another way we wouldn't reach through these that we made the open call so first we contacted people on ONGs or associations that we knew that had a connection to a certain location or space in Lisbon and surroundings just to make sure that they understood what we were asking for and we asked for help for them to spread the word and that's how we got our 21 youngsters actually we got 30 21 and I just share this with you because sometimes it's in the tiny details that our stereotypes show up when we had to select because we had too many applicants and too few places then we had to make a selection and we just asked for very simple things so it could be three paragraphs about why do I want to be part of this it could be three photos or it could be a video with three minutes at the most and people just chose different ways and suddenly the people that sent photos didn't write anything well we didn't ask for that and we got ourselves into a position that we were making decisions and we were giving priority to the people that explained very clearly why they were going to come and then suddenly someone in the middle of this said we're doing this all wrong again because we are again using the stereotype of the ones who can write very well are the ones who are going to pick so what about the other ones that chose a completely different way these are the ones we want to listen because these are the ones that usually don't show up so we just changed it again and we ended up with a very very diverse group which was really important most of these people had never come before to Gubankin and that was important well the other thing is another question that was there so how we did in the beginning so because of that because we knew that a lot of these people had no connection to the Gubankin maybe only with the name and the symbolic statues but not the real thing the first moment was also to connect with the institution because they were going to program not for another place but for this particular institution and you need to be aware of the constraints and the possibilities that this open and so they participated in most of the cultural events that were happening at the time so they get an idea of what was the actual programming we had they contacted with all the programmers inside the institution the stage director everyone connected to you know putting up out there some cultural offer for any kind of audience and then we invited people from other institutions and this was the time where they made the first decisions because they were the ones who picked up the other programmers they wanted to talk to so we could have kind of a round table of discussion about programming I guess I answered all the questions that were well Susanna thank you so much for that I think also this anecdote about the selection process is interesting because I mean you had to start the new listening phase very early on and also what you said before that too about involving the whole organization we hear a lot about how important it is to be holistic and the whole organization should be involved with the risks but I think one of the things you pointed out not only are you getting the best ideas that way but also you have more mobility that way kind of addressing those blocking forces I think that's certainly important to keep in mind if you all have time I would take at least a couple more questions from the chat if that's fine Inesh this is directly to you actually if an institution would like to implement this strategy would MAPPA be able to accompany and tutor this methodology actually answered Philippa by by writing yes of course all the partners of the project are willing and available for the implementation or the dissemination of the methodology but we will also have the blueprint as an open source right until the end of the year so the idea is that you can go both ways even though that we recognize that the present of the strange ingredient is important for the implementation of the methodology but our goal as a team is to have it as an open resource for anybody but of course MAPPA is more than available and very interested in that of course thank you so much and then here as well not so much a question but more of a reflection it says here thank you so much for these inspirations I think this process could especially help on one of the biggest challenges and changes for society and our institutions and that is to address climate action and I'm wondering especially with bringing youngsters in as was done Susanna I'm wondering if this conversation came into play perhaps also due to everything going online due to the pandemic if that came up at all yeah that was one of the subjects that I really wanted to make sure that we would address and for instance for the roundtables that you saw online they chose particularly people that were connected to this climate strike so they would have this voice presence as a discussion and they were demanding as in most of the other subjects but they were demanding that cultural institutions also made a stand and just take a position in these really crucial matters so that's something that we learned and for instance next year one of the major things we're going to work not only with these youngsters I'm hoping but as the modern art center it will be actually connected to ecology and all this climate change because I think museums have to be aware that we need to address in an artistic form but we need to address the current issues that are really concerning us not now, not only now as a generation but also in the future actually this isn't just another thing which is maybe something that we forget institutions have most of the times a mission and a mission statement and for us it was also very important and it was the way we got to be supported is that we knew that we were addressing very clearly all the mission that was behind the foundation role in Portuguese society and we even made sure that we were able to connect to other departments that were out of this project just because we knew that one of the strategic lines for the last few years was going and work with the younger generations in all in all departments so we knew that we were just choosing also a target audience that was at the center of our strategic plan and that makes it more relevant for the institution and sometimes that's a way to make it easier in all the process Yeah, absolutely I mean finding a way to really honor your mission statement but still be able to develop and move forward and also tackle some of these really difficult and pressing discussions and challenges for society is a lot for a cultural institution to take on and you know I think that's why programs such as this where we can lean on each other and learn from each other are so valuable Yeah I mean I think we could continue with this for quite some time but we're already I think 10 minutes over our time so unless any one of you has a final statement that they'd like to share then close for the day well thank you so much thank you all for being here thank you to Edeste Plus all around and yeah wishing you all a pleasant rest of your afternoon then Thank you so much Bye


hello everyone this is Amit Dan of amitdan.com you are watching one of the first travel videos on the sovereign hill playlist and today i am taking you back in time 200 years ago. sovereign hill in ballarat a small city from the 1850 gold rush in australia is preserved as it was back in those days. you will be able to go back in time and experience a gold mine city. experience time and life in this recreated gold township of the 1850s with me. in this episode i'll take you inside a boiler house in the gold city sovereign hill. these are two cornish boilers powered by wood-fired furnaces. steam from these boilers powered all engines of the mine and the bell-driven machinery in the sohokanvi workshop.


After doing a discovery based proteomics experiment, let us assume you have identified a protein of interest for which you do not know what is the possible role of that protein. You want to characterize that protein in much more detail, so possible way of doing that experiment could be you can do immunoprecipitation and identify that this protein of interest interacts with which other proteins. So, protein interaction networks could at least tell you the possible role that what that protein may have. In this slide immunoprecipitation followed by mass spectrometry have become a very powerful technique to do the protein interaction analysis. In today's lecture, we will have a demonstration by a research scholar who will show you how to perform immunoprecipitation mass spectrometry based experiment. Raisin or a bead support and we will conjugate our antibodies with it and those antibodies are definitely depends on your experiment, but they will be specific to a particular protein right and that protein you can trap on your antibody and then that protein can act as a bait and on it many of its interacting partners can get bound to right. So, what we will do is we will separate this whole complex. So, we will be doing a complex zone study or an interactome study using mass spectrometry. Though immunoprecipitation predominantly also the co immunoprecipitation is used in even in western blocks where you can use one protein and see whether it is interacting with another protein and you can use an cross antibody and check it. So, this also is a is basically how IP is conventionally used, but here we will be doing it how we will study it using like we in western block you can identify maybe one interacting partner, but using mass spectrometry you can identify a plethora of proteins from your complex. So, thereby I will be talking about understanding protein complexes and their interacting partners using IPMS. So, basically this is the workflow, so we will have a bead support majorly people use Cepherose beads, people use agarose beads also with protein A or protein G immobilized because protein A or G will bind to the FC region of the antibody. So, you will conjugate your antibody and then you will incubate this. So, in the protocol you will incubate your bead antibody conjugate for overnight, so that from your lysate you can trap the protein of interest. So, we will call this protein of interest which is bound to your antibody as the bait and then this bait will actually help you trap all the proteins the interacting partners of this protein. So, in we will take a case study today that is stat 3. So, stat 3 as you know is a part of the jack stat pathway and what if if you were interested to see that how which proteins are there potential interactors of stat 3. So, we will take an anti stat antibody and we will conjugate it with the bead and then stat 3 will let us assume that this yellow dot is stat 3. So, stat 3 will bind to the antibody and then all the interacting partners of stat 3 will we are expecting that it will come and bind to stat 3 and then what we can do. So, this is only one of the ways that you can do IP there are many ways to do it. So, people also directly use the antibody on the lysate and then they trap the complex. So, that can also be done. So, there are modifications of it, but in this case we will have the whole complex home like this and then we can run a simple SDS space, we can excise the bands and do an ingelization and give it to mass spectrometry analysis. So, what will happen you can exactly know which molecular regions you are looking at and you can go back to the database and you can see at your identified proteins on the mass spec and you can know how relevant are your hits whether at all their interacting partners of stat 3, but at least rather than doing a global proteomics like many of you are familiar with wherein you are identifying everything at least now you have just the way you can enrich your PTM you can enrich your sample to look at a particular complex right. So, this is the overall methodology there is an alternate way to do it in you can use this complex and use a buffer like something that has high pH like a glycine buffer what it will do this interaction will break the bead and the antibody interaction will break off and then you can do an in solution digestion and that you can subject to mass spectrometry analysis. So, overall this is the simple workflow of IPMS. So, protocol in brief what we do is first of all we will use the bead slurry and we will decant of the buffer. So, then the antibody mixture is incubated at 4 degrees for 1 to 4 hours. So, this is to ensure that the antibodies properly bound to the bead because that is very important that is what will pull. So, you are sort of doing a pull down essay we can also say that right and then you will centrifuge to get rid of any like things that are not bound. So, unbound things will be you will get rid of it and then you will wash the mixture using IP wash buffer. So, basically it is trace NACL and all those buffers they will keep your protein intact. And then, but at any point you must remember that for this to be successful you should not disrupt the interaction. So, all your steps you need to be very gentle you need not to you should not use something like a very harsh chemical like urea or anything that can denature your protein or even disrupt your interaction. So, that is something that you should keep in mind and then this lysate bead antibody conjugate this mixture you can keep for with a rotary gentle agitation at 4 degrees for overnight binding. So, now again it depends on your experiment you can plan it according if you know like if you have already verified the response using a western blot you will know how much of it of the protein of interest is in your sample. So, accordingly you can design if you if you think that it is too abundant and maybe you might not go for an overnight incubation. So, these things can be optimized. So, further the next day so it basically IP takes like a consecutive three days. So, you can use like a couple of days to get this done. So, then in the next day you will again wash it. So, again you are washing so as to not have those things that are just weekly bound or maybe as a carry over you just want to be sure that you are identifying only the complex of your interest and then you will remove it you will do an elution. So, again this step is optional you can directly load the whole thing in the gel. So, whatever it is it will come up and also you can use the whole complex and do an in gel in solutionization after the elution step. So, applications of IPMS so IPMS is very widely you know is being used now because conventional IP methods have also led to understanding of interactions protein-protein interactions. So, integrating with mass spectrometry just takes it to another level because now you you can do functional essays using mass spec as well and you are people there are many people who do targeted validation of proteins peptides using targeted proteomics for them as well. Their traditional biologist often raises a question that have you monitored the you know have you done a western laws have you done an Eliza. So, for them to if you have someone who wants to address both these crowds you can definitely use this technique. So, one of the preliminary studies that have come up is this by Mathias Olin group. So, they have given a proposal by which you can validate antibodies. So, here by on now onwards I will be talking about three major applications of IPMS. So, there are much many, but in this scope of lecture so application one of the applications I will be talking about is validation of antibodies using immunocapture technique. So, immunocapture is again IPMS because you are capturing the antibody of your interest followed by a mass spectrometry. Secondly, we will be talking briefly about an identifying protein-protein interaction that is interactomics. This is what the this is the demo session that you will be going for. And the third thing is enriching residues that are less in abundance and undergo iron suppression like phosphorylation or even ubiquitinylation and many other PTMS. So, because of that it is often very difficult to you know even if the if the post translational modification is very complex like a ubiquitinylation it is not easy to identify using a global proteomics approach. So, we can also use IPMS for enriching those residues like we can enrich the residues in we can ubiquitinylate the proteins and see which are the residues getting ubiquitinylated. We can use a tyrosine specific antibody which can bind to the tyrosine residues tyrosine phosphorylated residues and then we can do a mass spectrometry. So, thereby there are many things that you can do with this technique. And to the first application. So, in this proposal for validation of antibodies Mathias Olin group what they have proposed is that immuno capture followed by mass spectrometry is a very nice way to know whether your antibody specific or not. So, what they are aiming for is if your antibody is very specific to your target. So, if you bind it to a bead conjugate and then you do an immuno capture it should bind only to the protein of your intramus. If you do the washing if you follow the whole IP protocol what should you get is only your protein of interest and maybe a few interacting partners right. But so they have devised this statement that is an antibody can be considered specific if the top three peptides are derived from the expected target protein. So, if you are looking at an antibody. So, you have three antibodies say from vendor A's, AAPCAM, CST, Sigma. So, you want to know which one works the best for your experiment. So, before probably you are going to do something very you might use something very expensive or you have some sample which is very precious. So, before you proceed you might want to check how specific your antibodies. So, you can do an immuno capture and you can just see whether the top three peptides that are coming up after mass spectrometry are of the protein of your interest in this case stat 3. So, if that is coming you can be confident that whichever antibodies more specific will give higher number of peptides or the top hits would be from the protein of interest. So, in this way they have proposed that for validating an antibody they have proposed a panel of many studies and one of the studies they have proposed is immuno capture. So, the second application is studying protein-protein interaction or the typical interactomics. So, as you can see for any physiological process that you are studying be it in plants humans cancer or infectious disease. So, you often find out your list of targets does not involve one protein you always have something else along with it may be a transcriptional factor may be a protein or another family of proteins. So, it is never in isolation. So, exactly this is what we can achieve by doing an interactomic study in it. So, we will get to see the PPIs or protein-protein interactors. So, in this way we can map the whole interactome using IPMS. So, in the first slide I had mentioned that the protein of interest can be used as a bit in which the various interactors will come and bind to the bit and so, you can identify the direct interactors. So, if protein A is interacting with protein B. So, stat 3 is direct interactor say June or whatever binds to stat. So, now even you can identify proteins that are bound to B or June or whatever it is. So, you are not only identifying the direct interactors, but you are also identifying the co you know indirect interactors. Thereby you can map it and you can form a map of the interactome. Of course, this alone might not be the ultimate experiment because there are hundreds of other things that there can be some molecules that are weakly bound or the washing might not be efficient. So, you need to verify whatever interactors you are getting through alternative techniques, but this can definitely give you a big list of proteins which are potential interactors. So, the application 3 I spoke about is how to study low abundant residues using IPMS. So, again taking one of the case studies, so in we can enrich our samples in the residue that we are interested to look into. So, just the way phosphoproteomic study can be done using TiO2, but you would be aware of the TiO2 technique, it only enriches serine and trionine. You would hardly see any tyrosine residues because tyrosine residues A are very less in the whole proteome and B they are very transient in nature. Sometimes they are phosphorylated, sometimes they are not phosphorylated, so it is hard to get. So, what you can do is you can you can take an antibody, you can pull the tyrosine phosphorylated residues and you can perform an MS to be very confident that you are getting the tyrosine phosphorylated residues. Similarly, in one of the studies what they did is, so they studied ubiquitinilation using IPMS. So, this is the study by an Annie et al. So, they have done peptide level amino affinity enrichment and enhancing the ubiquitinilation site identification on individual proteins. So as you can see, first of all they have monitored the response or the ubiquitinilation using an anti-ubiquitin protein. So one of the steps before you proceed for IPMS, you should check the response or the number of residues present or the protein of interest using a western blood. And secondly, you can do your IP experiment followed by so you can use the antibody and then the bait protein and then do an IP and then you can run in the gel and then you can make fractions of it and directly subjected to mass spectrometry. So in this case what they did is, so ubiquitinilation involves glycine to lysine residues. So the ubiquitin attached to the lysine residues of the substrate protein. So what they have done is, they have enriched the sample using this KGG stretch. So they have actually tried to only enrich samples that have that yeah ubiquitinilation and then what they have seen is, they have seen in even in the mass spectrometry they can identify the sites in which the ubiquitinilation, so they can see the stretches of K and G. Thereby they can be confident that these are the, they have identified the proteins that are enriched in ubiquitinilation. So now today's case study, we will be doing a small IP experiment. Of course the whole stretch of experiments is beyond the scope of this course. But what we will see is, we will see that those small steps wherein you conjugate the bead with the antibody followed by the centrifugation and of course the lysate and antibody bead conjugation, how it happens in the live session. So what we will do is, we will, we have split this experiment into three stages. So stage one is, we have verified the stat 3 is present in the tissue lysate that we are probing with. So presence of stat 3 was verified using the western blot. And then the protein is present, we can have two inferences. Number one that we have stat 3 in the sample because it is coming up in the western blot and number two the antibody is very specific because it has not given multiple bands. So then we have moved on to the IP experiment wherein we have done, we have immobilized stat 3, like we have enriched, strapped stat 3 on the antibody and then we have done the whole IP experiment and we have excised the band and it has been subjected to mass spectrometry. So the stage 3 of the experiment is identification of the proteins, specifically the ones that has been enriched through IP. So we are not taking the whole sample, we are just taking those bands that have come specific and we are subjecting it to mass spectrometry. So you will get a list of proteins and you can then do bioinformatic analysis. So you can do a scoring based on string, you can string DB which is an open access software. There are other softwares as well and you can come up with a score and you can do other experiments to see that how the list of protein you are getting from mass spectrometer, how closely they are the interactors of your interest. And thereby you can form an interactome map. We will demonstrate to this technique in detail as well as provide you the brief protocol how to perform these experiments. So let us have this demonstration session. Hello students. So in continuation with the lecture where we learnt the theoretical aspects of IPMS that is immunoaffinity and immunoprecipitation based pull down followed by mass spectrometry. Today we will see what are the steps in which way we can perform this experiment and how to do that for your biological problem. So first of all just to brief you a little bit about the steps. So majorly in the immunoprecipitation based mass spectrometry we will be doing the pull down of protein of whose interacting partners you are interested to look into. So for example let's say protein say S108N. So you want to learn what are the interacting partners of S108N. So for that first of all you need to have an antibody which is specific to S108N. So for that we need the antibody of the protein of interest whose interacting partners you will be finding out. So here first of all we will take the bead. So this is protein A or protein G beads you can take. So basically the protein A beads have the protein A which is derived from bacteria and it is highly specific and it binds to the FC region of the antibody. In that way we will conjugate the antibody of interest with the bead that is present in your tube. So first of all we will prepare an bead antibody conjugate and then because the antibody is specific to the protein of interest we will try to pull down that protein from your biological sample. So we will perform the experiment in three steps. Number one we will prepare the antibody bead conjugate. Number two we will be doing the pre clearing of the lysate. So for example you want to look into a specific cell line or patient derived tissue or tumor and you want to isolate the protein of interest. So for that we will do the we will first take the lysate of your sample of interest. So this lysate contains thousands of proteins and you do not want to have a very high background. So first we need to do a step which is called the pre clearing of lysate. And then the last step is using the antibody and bead conjugation the mixture where the antibody is already bound to the bead and using this pre clearing lysate which is the lysate which is clear of all the noise or all the background proteins that we will incubate overnight and this is the way we will actually try to get all the interacting partners together in a big complex which we will subsequently run in a SDS gel and then we will do the gel excision followed by the mass spectrometry. So as you can see we have the beads here. So first we have to add washed buffer in these beads. So usually what we will do is we will usually use the pre chilled washed buffer. So this buffer is already chilled. Why this is done is because the beads need to get like activated prior to the binding with the antibody. So the once you put the so you just give it a little bit of a tap and once you put the beads and the buffer so the beads will swell after some time and then you can perform a centrifugation step followed by which we can put the antibody. So we will just use we will just place it in a container and we will gently rock it for some time so that the buffer is uniformly distributed between the beads. So once it is mixed properly you can also again do a gentle tap and do an inversion so that there is nothing like there are no beads that are not in touch with the buffer and then we can do a centrifugation. So now that your beads are activated you can get rid of the washed buffer put some fresh washed buffer and in this and in this you can now add the antibody. So usually 2 to 5 microgram of antibody is usually used but then it depends on the protein of interest and how specific your antibody is. You can use monoclonal antibody as well as polycronal antibody. So for example if you are trying to pull down a protein which has a many of the family members like for example if you are pulling down something like anxin so there are many anxin proteins that are present in the human or mammalian tissues anxin A1, A6, A2 so no matter how specific your antibody is these all all the members of these protein will definitely cross react. So according to the protein that you want to select you need to optimize how much time you want to like keep this binding solution so you can go either for an overnight binding at 4 degrees or you can do a binding for 2 to 4 hours depending on your experiment. So now what we will do is we will add the antibody to the activated beads. So now the bead is already activated using the wash solution to which we will add the antibody. So after adding the antibody it is not advised that you do a very vigorous shaking or mixing so you just gently tap it so that everything is properly mixed and then you again place it in a container and put it on a locker. So either you can leave it for 2 to 4 hours or you can do this step overnight. Similarly for the lysate you can do a same step wherein you incubate instead of the antibody you incubate the lysate with the bead. So here there is already a pre-cleared lysate and after this step is done your beads are already containing the antibody of your interest we will now put the pre-cleared lysate into the antibody bead conjugate. So here the beads have your antibody and now we will add the pre-cleared lysate.  After the incubation you can see that now the beads which are conjugated with the antibody will bind to the protein of your interest from the lysate from the pre-cleared lysate. So after this what will happen is you will already have the antibody bound to the protein of your interest and ideally from the lysate all are the proteins which are interacting with the protein of your interest will stick to it and form a complex. So what we will do is we will discard the flow through we will give it a brief spin we will store the flow through in a separate tube and now we will perform washing. Then why do we have to do washing because if you want to reduce the background that is if you if you consider this mixture it has lots of proteins which are also binding to the antibody bead conjugate which is also which could be not very strong interactors of your protein. So the moment we add buffer we will give it a gentle mix so what will happen is all these loosely bound proteins will get washed away. So you will usually we perform a washing for like 2 to 5 times again depending on the protein of your interest and your specificity of the antibody you can optimize these parameters. So we will gently mix the buffer with the complex and then we will give it a gentle stir and then again do the centrifugation. After giving a gentle spin will again store the flow through in the separate tube set will be repeated for 2 to 3 times you can even go up to 5 times depending on the specificity of your antibody and then what we will do is so now we only need to look at the bead and the conjugated complex. So what we will do is there are two ways to go about it so either you can use an illusion buffer which has a lesser pH that is you can use Trisglycin as a buffer. So what it will do is it will disrupt the bonds now it will disrupt the linkage and it will elute out the complex from the bead. So we will use the illusion buffer and again give it a gentle tap after that we will again put it in the rocker and leave it for few minutes. Make sure this is not too harsh like you don't do it do a vigorous mixing or shaking or even in this rotator because the you don't want to lose the interacting partners that have already bound to the protein of your interest. So with this now we are sure that we have got rid of all the background that might interfere with our study and now we have only the antibody bound to the protein of interest and the interacting partners after the illusion step even the bond between the antibody and the proteins of interest starts getting disrupted. So what we will do is we will again use a little bit of IP buffer and will perform a spin. Once this is done you have to take the supernatant and put it in a fresh vial. So this supernatant now contains your protein of interest as well as all its interacting partners. So there can be two ways by which you can go for the protein identification. Number one you can go for the insolution digestion of this elute. Number two you can perform an ingel digestion. So here you can see when you run the protein in a gel you will get different bands and now you don't know what is what. So what happens is various proteins that are present in the mixture will get separated in the SDS. So what we will do is we will do the ingel digestion. So we will cut the gel and we will make small small pieces depending on the number of fractions you want to do. For example I will cut this lane into four pieces. So this you can see there is a prominent band here. This looks like the protein of interest and probably many of the proteins that have become formed a complex with it. So this will cut into further smaller fractions will take the band that has been excised from the gel and will perform an ingel digestion. So here this you can see this small band may have hundreds of proteins which are potentially the interacting partners of the protein of interest against which you had used the antibody. So after this ingel digestion would be done and you will elute the peptides out. So this is the digested peptide and now we will inject it for mass spectrometric analysis. To summarize our entire experiment we have now performed immuno precipitation of a protein of interest from mammalian cells. So we have taken the cell lysate and we have pre-cleared it using beads and similarly in the another section of it what we have done is we have conjugated an antibody specific to the protein with the bead. So both of these steps were done and then the mixture of the antibody bead conjugate was incubated with the lysate. The protein of interest gets captured by the antibody and all its interacting partners also binds to that protein. So that we can get a big complex or a complexome or we can get the interactome that is present in the cell lysate. So after that what we did is we ran the sample in the gel and we excise the bands and this bands would now cut into small fractions and were digested. After digestion we have separated the peptide and this peptide would now be injected into the mass spectrometry to see what are the proteins, what are the other proteins that have interacted with our protein of interest. The advantage of coupling a very traditional method like immunoprecipitation with something advanced like mass spectrometry is that you get best of the both worlds that is while antibody based detection can detect even a very low abundant protein mass spec can help you identify that protein as well as several other proteins which are potentially the interacting partners of the protein of interest. Therefore IPMS is a very promising approach to look into mechanistic details of how a cell performs, how a signaling cascade performs and hence it is emerging as a very prominent tool in the interactome studies. So as you saw I already briefed you about the experimental workflow of IPMS that is how we perform the immunoprecipitation using the antibody. After the process is done we also saw that we can perform ingel digestion as well as we can go for an insolution digestion of the precipitated complex and now when that is done what we have to do is we have to now submit the samples for mass spectrometry analysis. So now we will be injecting our sample in the mass spectrometer. So now the sample will be run using the LC gradients and the mass spectrometer and we will be possibly able to identify the interacting partners of our protein of interest against which the antibody was used. Therefore IPMS gives a very nice approach to identify not only just one protein but also its interacting partners and not only that one of the advantages it has over traditional immunoprecipitation approaches while immunoprecipitation mostly is followed by a western blot in which another counter antibody is used to see whether you can see the protein of interest binding to other interacting partners there are issues related to antibody specificity and cross reactivity. In this case whatever has been pulled down by the antibody and whatever has been bound to the the bait or that is the protein of interest so the bait and the praise can be characterized very confidently using the mass spectrometry and in the next lecture you will learn in detail how this approach works. Thank you. Hope now you are all familiar with the entire workflow of immunoprecipitation followed by mass spectrometry based experiment. You may have realized that it is a good way to characterize protein complexes and their response to regulatory mechanisms. The IP experiment allows hundreds of proteins to be identified in a single experiment however it is also important to keep in mind that a majority of proteins identified in this experiment may also be non-specific binders. And now you need to do further investigation about from the possible list of interactors how many of those could be the real or direct interactors or how many could be just the sticky proteins who are coming along with the interactors of interest. Researchers are now looking ways to improve the accuracy of identifying these interactors which could be drawn from the IP experiment. However the way mass spectrometry technologies have now become more robust and easy to operate it is now not very difficult to perform immunoprecipitation followed by mass spectrometry experiments. We will give you some basics of mass spectrometry and how to perform these experiments in next class. Thank you.