Determining protein stability in cell lysates by pulse proteolysis and Western blotting
AUTOR(ES)
Kim, Moon-Soo
FONTE
Wiley Subscription Services
RESUMO
Proteins require proper conformational energetics to fold and to function correctly. Despite the importance of having information on conformational energetics, the investigation of thermodynamic stability has been limited to proteins, which can be easily expressed and purified. Many biologically important proteins are not suitable for conventional biophysical investigation because of the difficulty of expression and purification. As an effort to overcome this limitation, we have developed a method to determine the thermodynamic stability of low abundant proteins in cell lysates. Previously, it was demonstrated that protein stability can be determined quantitatively by measuring the fraction of folded proteins with a pulse of proteolysis (Pulse proteolysis). Here, we show that thermodynamic stability of low abundant proteins can be determined reliably in cell lysates by combining pulse proteolysis with quantitative Western blotting (Pulse and Western). To demonstrate the reliability of this method, we determined the thermodynamic stability of recombinant human H-ras added to lysates of E. coli and human Jurkat T cells. Comparison with the thermodynamic stability determined with pure H-ras revealed that Pulse and Western is a reliable way to monitor protein stability in cell lysates and the stability of H-ras is not affected by other proteins present in cell lysates. This method allows the investigation of conformational energetics of proteins in cell lysates without cloning, purification, or labeling.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2771307Documentos Relacionados
- Differentiation of Larva Migrans Caused by Baylisascaris procyonis and Toxocara Species by Western Blotting▿
- Molecular Markers in Acute and Chronic Phases of Human Toxoplasmosis: Determination of Immunoglobulin G Avidity by Western Blotting
- Comparative Molecular Analysis of the Abnormal Prion Protein in Field Scrapie Cases and Experimental Bovine Spongiform Encephalopathy in Sheep by Use of Western Blotting and Immunohistochemical Methods
- In vitro production of bacitracin by proteolysis of vegetative Bacillus licheniformis cell protein.
- Analysis of western blotting (immunoblotting) technique in diagnosis of congenital syphilis.