title: Immunoblotting of hydrophobic integral membrane proteins.
abstract: For diagnosis and research purposes it is frequently desirable to measure by immunoblotting small amounts of proteins in complex mixtures such as tissue biopsy homogenates. Standard immunoblot procedures that give excellent results for soluble proteins unexpectedly gave low and irreproducible signals with some hydrophobic membrane proteins. We found that this was due to inefficient electrophoretic transfer to nitrocellulose, which could be corrected by modification of the transblot buffer. Hydrophobic integral membrane proteins of peroxisomes as well as other rat and human liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. The nitrocellulose-bound proteins were detected both by staining and by immunoblotting with an antiserum against the 22-kDa integral membrane protein of peroxisomes plus 125I-labeled protein A. A modified transblot buffer with 0.7 M glycine and 25 mM Tris (pH 7.7) but no methanol allowed use of a much shorter transfer time and strikingly improved the electrophoretic transfer of membrane proteins such that a peroxisomal integral membrane protein could be easily detected in human liver biopsy homogenates.
authors.affiliation: Rockefeller University, New York, New York 10021.
authors.forename: G M
authors.initials: GM
authors.lastname: Small
authors.forename: T
authors.initials: T
authors.lastname: Imanaka
authors.forename: P B
authors.initials: PB
authors.lastname: Lazarow
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007158:Immunologic Techniques; D008099:Liver / Q000032:analysis; D008565:Membrane Proteins / Q000302:isolation & purification*; D008830:Microbodies / Q000032:analysis; D051381:Rats
chemical_list: D008565:Membrane Proteins