High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
High-molecular-weight proteins often blot onto nitrocellulose membranes poorly following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting in low levels of detection on immunoblots, and hence difficulty in analyzing rare proteins. Moreover, optimizing conditions for the transfer of high-molecular-weight proteins to nitrocellulose frequently results in the inefficient transfer or loss of lower molecular weight proteins. This problem is particularly vexing during the analysis of large proteins which may be processed to one or more smaller biologically active forms. Using radiolabeled protein standards and phosphorimaging technology, we have quantitated the efficacy of many different protein gel electrophoresis and blotting protocols. Here we report novel gel and blotting conditions which significantly improve the transfer and retention of high-molecular-weight proteins, without sacrificing the efficient transfer of lower molecular weight proteins. Using this newly described procedure, we have detected a rare 500-kDa protein in immunoblots which was previously not detectable with any of the commonly used blotting procedures. Since the improved conditions offer increased sensitivity across a spectrum of protein sizes, they should be widely applicable.
Bolt
M W
MW
Division of Biological Sciences, University of Missouri, Columbia 65211, USA.
Mahoney
P A
PA
D015153:Blotting, Western / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D002021:Buffers; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate
D002021:Buffers; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D003101:Collodion