Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.
A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.
Abeyrathne
Priyanka D
PD
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Lam
Joseph S
JS
D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods*; D002021:Buffers; D003101:Collodion / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008070:Lipopolysaccharides / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D015203:Reproducibility of Results
D001426:Bacterial Proteins; D002021:Buffers; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D003101:Collodion