A high temperature transfer procedure for detection of G protein gamma subunits by immunoblotting.
The production of antibodies against synthetic peptides derived from amino acid sequences common or unique to a particular protein(s) is an important tool in the identification of structurally related members of an ever-increasing number of protein families. The successful use of anti-peptide antibodies requires that the protein(s) of interest be properly transferred and fully denatured prior to detection by Western-type immunoblotting. In this paper, we demonstrate that conventional transfer procedures are not successful in presenting the G protein gamma subunits in a suitable state for immunodetection. We describe a high temperature (70 degrees C) transfer procedure that results in a more than 20-fold enhancement in the sensitivity of immunodetection of the various G protein gamma subunits. The effect of high temperature transfer could not be duplicated by including 0.2% SDS in the buffer during transfer to nitrocellulose, or by baking or autoclaving the nitrocellulose after transfer. Thus, high temperature transfer is a powerful procedure for enhancing immunoblot detection of protein(s) that may be resistant to denaturation and/or subject to renaturation during the transfer and/or binding to nitrocellulose.
Robishaw
J D
JD
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Balcueva
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D003101:Collodion; D000939:Epitopes / Q000737:chemistry; D005069:Evaluation Studies as Topic; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D006358:Hot Temperature; D015151:Immunoblotting / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate
D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D012967:Sodium Dodecyl Sulfate; D003101:Collodion; D019204:GTP-Binding Proteins