Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots.
When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself (C.B. Klee, T. H. Crouch, and M. H. Krinks, 1979, Proc. Natl. Acad. Sci. USA 76, 6270-6273) permitted the detection of only very high affinity Ca(2+)-binding proteins.
Garrigos
M
M
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Associé à l'Université Pierre et Marie Curie Paris (VI), Gif-sur-Yvette, France.
Deschamps
S
S
Viel
A
A
Lund
S
S
Champeil
P
P
Møller
J V
JV
le Maire
M
M
D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D002155:Calsequestrin / Q000032:analysis; D004586:Electrophoresis / Q000379:methods; D005782:Gels; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods; D007700:Kinetics; D008567:Membranes, Artificial; D008121:Nephropidae; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate
D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D005782:Gels; D008567:Membranes, Artificial; D012967:Sodium Dodecyl Sulfate; D000251:Adenosine Triphosphatases; D002118:Calcium