Application of immunological methods for the visualization of PK after IEF in ultrathin layers

Immunological methods are recommended for protein visualization after IEF on account of their high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase- and 125I-labelled antibodies to investigate microheterogeneity of pyruvate kinase (PK) from human red blood cells and could show a PK pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization on the polyacrylamide gel. Beside Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques especially after isoelectric focusing in ultrathin gels.