Horizontal semi-dry electroblotting for the detection of the low density lipoprotein receptor in solubilized liver membranes.
A high efficiency transfer of the low density lipoprotein (LDL) receptor proteins from polyacrylamide slab gel onto immobilizing nitrocellulose membranes using the horizontal semi-dry electrophoretic system is described. The transfer of the LDL receptors from solubilized rat liver microsomes was performed between two graphite plate electrodes in a continuous buffer system containing methanol and sodium dodecyl sulfate. The protein transfer was achieved in only 150 min at a constant current of 0.8 mA/cm2 at room temperature with very low Joule heat development. The homogeneous electric field yield between the two electrode plates produced a satisfactory transfer of the LDL-receptor protein band in spite of its high molecular weight, and only few protein traces remained in the polyacrylamide gel after blotting. This improved method allows a rapid and quantitative transfer of the LDL receptors without protein denaturation, since the specific binding activity of the blotted receptor is retained as demonstrated by ligand-blotting and immunoblotting.
Himber
J
J
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
D000818:Animals; D001345:Autoradiography; D003101:Collodion; D004492:Edetic Acid / Q000494:pharmacology; D004560:Electricity; D004591:Electrophoresis, Polyacrylamide Gel; D004997:Ethinyl Estradiol / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000737:chemistry* / Q000187:drug effects; D051381:Rats; D011973:Receptors, LDL / Q000032:analysis*
D011973:Receptors, LDL; D004997:Ethinyl Estradiol; D003101:Collodion; D004492:Edetic Acid