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Two‐dimensional gel electrophoresis of actin‐binding proteins isolated by affinity chromatography from human skeletal muscle
Author(s) -
Coumans Joëlle V. F.,
HumpherySmith Ian,
dos Remedios Cristobal G.
Publication year - 1997
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/elps.1150180709
Subject(s) - actin , deoxyribonuclease i , actin binding protein , skeletal muscle , affinity chromatography , agarose , biochemistry , gel electrophoresis , chemistry , biology , biophysics , cytoskeleton , actin cytoskeleton , dna , enzyme , cell , anatomy , base sequence
In muscle cells actin exists as a mixture of monomeric (G‐actin) and filamentous actin (F‐actin) and ionic conditions strongly favor the formation of F‐actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G‐actin, the so‐called G‐actin‐binding proteins (G‐ABPs). We have coupled monomeric actin to divinylsulphone‐activated agarose (Mini‐Leak) to isolate G‐ABPs in human skeletal muscle. Eluted proteins were analyzed by two‐dimensional gel electrophoresis (2‐DE), which shows that some proteins are selectively retained. Deoxyribonuclease I (DNase I) is known to bind residues at the “pointed end” of actin (subdomains 2 and 4) with a high affinity. When DNase I is bound to the actin Mini‐Leak before applying the skeletal muscle extract, the 2‐DE gels of the eluted proteins reveals differences when compared to gels of proteins eluted from actin‐Mini‐Leak and DNase I‐Mini‐Leak affinity columns. This strategy should detect ABPs which bind to sites other than the DNase I‐binding site and some may prove to be novel.