Enzymatic detection of native and derivatized horseradish peroxidase in sodium dodecyl sulfate polyacrylamide gels.
We developed procedures for the restoration of peroxidatic activity in native horseradish peroxidase (HRP) and HRP conjugated to wheat germ agglutinin (WGA-HRP) following electrophoresis in SDS-polyacrylamide gels (SDS-PAGE). After extraction of SDS with isopropanol from gels containing HRP and WGA-HRP, the peroxidatic activity in these probes could be demonstrated by tetramethylbenzidine (TMB) chemistry. This procedure also showed HRP enzyme activity in electrophoresed tissue homogenates containing HRP. Both free HRP as well as WGA-HRP preparations contain several molecular weight species that display peroxidatic activity. These findings are important for cell biological studies utilizing these substances as molecular probes. The procedures described here should be useful for the analysis of the enzymatically active molecular forms of these frequently used markers in vitro and in vivo.
D000818:Animals; D001560:Benzidines; D001692:Biological Transport; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000032:analysis* / Q000378:metabolism; D037102:Lectins; D008970:Molecular Weight; D010544:Peroxidases / Q000032:analysis*; D051381:Rats; D012160:Retina / Q000201:enzymology; D013477:Superior Colliculi / Q000201:enzymology; D014909:Wheat Germ Agglutinins
D001560:Benzidines; D037102:Lectins; D014909:Wheat Germ Agglutinins; C021758:3,3',5,5'-tetramethylbenzidine; D006735:Horseradish Peroxidase; D010544:Peroxidases