Urea gradient/SDS-PAGE; a useful tool in the investigation of signal transducing G proteins.
We describe an updated and improved protocol to perform urea gradient/SDS-PAGE in which proteins are electrophoresed through 9% polyacrylamide gel slabs in the presence of a linear 4 M to 8 M gradient of urea using Laemmli's separation buffers. We provide examples of this technique to separate PTX labeled G protein alpha subunits, as well as unlabeled alpha and beta subunits of G proteins. Applications of the technique are exemplified in which (1) the chromatographic separations of G proteins in DEAE-Toyopearl and MonoQ columns are compared, (2) the complexity of PTX substrates expressed in human erythrocytes, bovine brain, dog ventricle, FRTL-5 cells, HIT cells, GH4C1 cells and RIN cells are compared, and (3) the polypeptide composition of G protein beta gamma subunits, as expressed in several tissues and found in three distinct G proteins from a single cell population, are analyzed.
Codina
J
J
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Grenet
D
D
Chang
K J
KJ
Birnbaumer
L
L
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002853:Chromatography, Liquid; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D008566:Membranes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014508:Urea; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D014508:Urea; D019204:GTP-Binding Proteins