High-resolution slab gel isoelectric focusing: methods for quantitative electrophoretic transfer and immunodetection of proteins as applied to the study of the multiple isoelectric forms of ornithine decarboxylase.
A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric forms of ornithine decarboxylase. Separation of proteins at this high level of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or different commercial sources, differed significantly in their protein binding capacity. Ampholytes bound to proteins interfered both with the electrophoretic transfer of proteins from the gel to immunoblotting membranes and with the ability of antibodies to interact with proteins on the immunoblotting membranes. Increasing the amount of protein loaded into a gel lane also decreased the efficiency of the electrophoretic transfer and immunodetection. To overcome these problems, both gel washing and gel electrophoretic transfer protocols for disrupting the ampholyte-protein binding and enabling a quantitative electrophoretic transfer of proteins were developed. Two gel washing procedures, with either thiocyanate or borate buffers, and a two-step electrophoretic transfer method are described. The choice of which method to use to optimally disrupt the ampholyte-protein binding was found to vary with each lot of ampholytes employed.
Reddy
S G
SG
Department of Pharmacology, University of Texas Medical School at Houston 77225.
Cochran
B J
BJ
Worth
L L
LL
Knutson
V P
VP
Haddox
M K
MK
D000665:Ampholyte Mixtures; D007118:Immunoassay; D015151:Immunoblotting; D007525:Isoelectric Focusing / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis*; D009955:Ornithine Decarboxylase / Q000032:analysis*; D011506:Proteins / Q000032:analysis*
D000665:Ampholyte Mixtures; D007527:Isoenzymes; D011506:Proteins; D009955:Ornithine Decarboxylase