Horizontal semi-dry electroblotting for the detection of the low density lipoprotein receptor in solubilized liver membranes
✍ Scribed by Dr. Jacques Himber
- Publisher
- John Wiley and Sons
- Year
- 1993
- Tongue
- English
- Weight
- 486 KB
- Volume
- 14
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
the detection of solubilized liver
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 mAlcm2 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.