Antibody recognition of calcium-binding proteins depends on their calcium-binding status.
Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.
Winsky
L
L
Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-1266, USA.
Kuźnicki
J
J
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D064092:Calbindin 1; D064032:Calbindin 2; D064026:Calbindins; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D005557:Formaldehyde; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D010320:Parvalbumins / Q000737:chemistry / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; C576500:Calb1 protein, rat; C576512:Calb2 protein, rat; D064092:Calbindin 1; D064032:Calbindin 2; D064026:Calbindins; D002135:Calcium-Binding Proteins; D007106:Immune Sera; D010320:Parvalbumins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D005557:Formaldehyde; D002118:Calcium