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Modified immobilized pH gradient gel strip equilibration procedure in SWISS‐2DPAGE protocols
Author(s) -
Yan Jun X.,
Sanchez JeanCharles,
Rouge Veronique,
Williams Keith L.,
Hochstrasser Denis F.
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:4/5<723::aid-elps723>3.0.co;2-q
Subject(s) - chromatography , chemistry , iodoacetamide , immobilized ph gradient , alkylation , reagent , cysteine , mass spectrometry , matrix assisted laser desorption/ionization , dispersity , adduct , polyacrylamide gel electrophoresis , desorption , isoelectric focusing , biochemistry , adsorption , organic chemistry , enzyme , catalysis
In the present paper we report a revised protocol for immobilized pH gradient (IPG) gel strip equilibration involving a procedural modification between the first‐ and second‐dimensional separation in both analytical and preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). By changing the pH of the equilibration buffer (pH 8.0), the concentration of alkylating reagent (125 m M iodoacetamide) and the time of incubation (15 min), it has been possible to achieve increased cysteine (Cys) alkylation to completion with only one adduct of carboxyamidomethyl‐Cys formed. Importantly, the modification does not alter the 2‐D proteome patterns and therefore maintains the integrity of the existing SWISS‐2DPAGE entries. Results are presented for comparative analyses using human plasma, and for Cys analysis of human albumin to illustrate the advantages of the improved protein reduction and Cys alkylation. The modified step of IPG gel strip equilibration will assist protein digestion for matrix‐assisted laser desorption/ionisation ‐ time‐of‐flight ‐ mass spectrometry analysis, and make Cys quantitation possible without further in‐gel or on‐blot alkylation.