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 immunoblotting. Traditional methods for extracting cellular proteins by homogenization or detergent solubilization usually produce protein samples that are viscous (due to the presence of DNA) and prone to degradation due to the presence of endogenous protease activity. We have developed a method that involves solubilization of cells with sodium dodecyl sulfate (SDS), precipitation of proteins with trichloroacetic acid (TCA) with special physical exclusion of DNA aggregate and reconstitution of precipitated proteins with Tris base. Protein samples prepared by this method contain little DNA, making them ideal for long-term storage. The solubilized total protein extracts are fully compatible with protein assay, gel electrophoresis and Western blotting. When compared to protein extracts from a homogenization method, those from the TCA method showed an identical total protein staining pattern on SDS polyacrylamide gel electrophoresis and contained distinct cellular proteins recognized by many monoclonal and polyclonal antibodies tested (including anti-actin, spectrin, protein kinase C (alpha), talin and spectrin) on Western blots.
Wang
K K
KK
Department of Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA. wangk@aa.wl.com
Posner
A
A
Hajimohammadreza
I
I
D000199:Actins / Q000302:isolation & purification; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002478:Cells, Cultured / Q000737:chemistry; D002540:Cerebral Cortex / Q000166:cytology*; D003598:Cytoskeletal Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D011493:Protein Kinase C; D051381:Rats; D013049:Spectrin / Q000302:isolation & purification; D016608:Talin / Q000302:isolation & purification; D014238:Trichloroacetic Acid; D014407:Tumor Cells, Cultured / Q000737:chemistry
D000199:Actins; D003598:Cytoskeletal Proteins; D016608:Talin; D013049:Spectrin; D014238:Trichloroacetic Acid; D011493:Protein Kinase C