Western blotting and immunochemical detection of histones electrophoretically resolved on acid-urea-triton- and sodium dodecyl sulfate-polyacrylamide gels.
We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mM acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mM Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated.
Delcuve
G P
GP
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Davie
J R
JR
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001943:Breast Neoplasms; D002460:Cell Line; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry; D005260:Female; D006657:Histones / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D007202:Indicators and Reagents; D008970:Molecular Weight; D011092:Polyethylene Glycols; D012967:Sodium Dodecyl Sulfate; D014508:Urea
D006657:Histones; D007202:Indicators and Reagents; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D014508:Urea