About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis.
The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels.
CEA-Laboratoire de Bioénergétique Cellulaire et Pathologique, DRDC/BECP Grenoble, France.
Sylvie
S
Luche
Hélène
H
Diemer
Chistophe
C
Tastet
Mireille
M
Chevallet
Alain
A
Van Dorsselaer
Emmanuelle
E
Leize-Wagner
Thierry
T
Rabilloud
D000478:Alkylation; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005782:Gels / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014867:Water
D004220:Disulfides; D005782:Gels; D008301:Maleimides; D010455:Peptides; D013438:Sulfhydryl Compounds; D014867:Water; C043592:maleimide; D003553:Cystine; D006861:Hydrogen Peroxide; D003545:Cysteine; D004229:Dithiothreitol; D007460:Iodoacetamide