TOTAL PROTEIN EXTRACTION FROM CULTURED-CELLS FOR USE IN ELECTROPHORESIS AND WESTERN BLOTTING

Experimentation with cultured cells often requires analyzing cellular protein extract by gel electrophoresis and immuno-blotting. Traditiona l methods for extracting cellular proteins by homogenization or deterg ent solubilization usually produce protein samples that are viscous (d ue to the presence of DNA) and prone to degradation die to the presenc e of endogenous protease activity. We have developed a method that inv olves solubilization of cells with sodium dodecyl sulfate (SDS) precip itation of proteins with trichloroacetic acid (TCA) with special physi cal exclusion of DNA aggregate and reconstitution of precipitated prot eins with Tris base. Protein samples prepared by this method contain l ittle DNA, making them ideal for long-term storage. The solubilized to tal protein extracts are fully compatible with protein assay, gel elec trophoresis and Western blotting When compared to protein extracts fro m a homogenization method, those from the TCA method showed an identic al total protein staining pattern on SDS polyacrylamide gel electropho resis and contained distinct cellular proteins recognized by many mono clonal and polyclonal antibodies tested (including anti-actin, spectri n, protein kinase C (alpha), talin and spectrin) on Western blots.