AMS alkylation  From OpenWetWare  Jump to: navigation, search
Goal: Disulphide bridges detection by AMS alkylation.
References: Rodríguez, I. et al, 2006 (PMID: 16537584) with
modifications. Procedure: AMS
(4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid,
Molecular Probes) alkylates free cysteines, adding 0.5 kDa.
Therefore, alkylation of reduced and non-reduced sample led to
different electrophoretic size.  1. You need four different
samples:   i) Non treated  ii) Reduced (this control is
essential since reduced protein may change mobility).  iii)
Alkylated (only free cys will shift electrophoretic mobility).
iv) Reduced and alkylated (all cys bounded or not will shift
electrophoretic mobility). Eventually you may prepared a oxidize
control, pre-treating the sample with 20 mM H2O2.  This protocol
can be used for bacteria or eukaryotic cells. For reducing
samples, add 200 mM DTT and let the culture go on for 30 min
extra.  2. Extracts preparation: -Wash the cells with ice cold
PBS. -Precipitate intac