Electrotransfer of proteins after isoelectric focusing (with or without urea) on fabric-reinforced, immobilized pH gradient gels.
In the present article a procedure is described which combines the horizontal isoelectric focusing (IEF) of proteins on fabric-reinforced polyacrylamide gels with the subsequent electrophoretic transfer of the proteins to nitrocellulose or Immobilon. The application of a carrier material that is permeable for current and molecules and that serves as a physical support of the IEF gel is one of the central prerequisites for the method to work. Moreover, it is important to fix the pH gradient topographically by the use of Immobilines mainly in order to avoid distortion of the protein pattern during the electrotransfer (Western blot). The Western blot can be performed either in the submerse or in the so-called "semi-dry" blotting system. Our procedure is compatible with IEF protocols employing buffer systems with or without urea. The efficacy of our method is demonstrated by the IEF and Western blotting of several known marker proteins.
Knierim
M
M
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Buchholz
J
J
Pflug
W
W
D000180:Acrylic Resins; D003101:Collodion; D004563:Electrochemistry; D005782:Gels; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D011506:Proteins / Q000032:analysis*; D014508:Urea
D000180:Acrylic Resins; D005782:Gels; D011506:Proteins; C016680:polyacrylamide gels; D014508:Urea; D003101:Collodion