Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate.
A method for the immunoelectrophoretic analysis of both hydrophilic and hydrophobic proteins from whole-cell extracts solubilized with 2% (w/v) sodium dodecyl sulfate (SDS) is described. For rocket immunoelectrophoresis, Triton X-100 is added to the sample before electrophoresis to sequester non-protein-bound SDS, and polyethylene glycol (PEG) is added to the antibody gel to enhance precipitin formation. With the optimal ratio of Triton X-100 to PEG, the quantitative determination of 5 ng of protein is possible. The SDS-solubilized sample can also be analyzed by crossed immunoelectrophoresis using SDS-polyacrylamide gels in the first dimension and antibody-containing agarose gels in the second. The best results are obtained when intermediate gels without nonionic detergents are used and when ionic detergents are omitted from the cathodal gel. Precipitin peaks of high quality, reproducibility, and without artifacts are obtained using antibody concentrations 5- to 50-fold lower than with other crossed-immunoelectrophoresis procedures.
D002696:Chlamydomonas / Q000032:analysis; D003432:Cross-Linking Reagents; D007122:Immunoelectrophoresis / Q000379:methods; D007123:Immunoelectrophoresis, Two-Dimensional / Q000379:methods; D010940:Plant Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility
D003432:Cross-Linking Reagents; D010940:Plant Proteins; D011506:Proteins; D012967:Sodium Dodecyl Sulfate