Inclusion of dimethyl sulfoxide-dissolved protease inhibitor cocktail in the lysis buffer cuts down the number of proteins resolved by two dimensional gel electrophoresis
P P, M.; Mundayoor, S.
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Two dimensional gel electrophoresis (2DE) resolves a mixture of proteins based on both isoelectric point and molecular weight of the individual proteins. Even when we followed a standard protocol for 2DE we got lesser number of proteins focused especially in the basic region of the IPG strip. Since the common troubleshooting measures did not solve the problem we replaced the protease inhibitor cocktail in the lysis buffer with PMSF which resulted in an ideal protein map following 2DE. We also found that the presence of the cocktail results in skewing of the mass spectra of a purified protein which eventually resulted in incorrect identification of the protein by MASCOT search. Later we found that dimethyl sulfoxide, the solvent of protease inhibitor cocktail also resulted in focusing of lesser number of proteins. Addition of 1% dimethyl sulfoxide to bovine serum albumin resulted in lesser sequence coverage for the protein by LC-MS/MS. Also, dimethyl sulfoxide was found to decrease the intensity value of the dominant peptide in a fraction when MALDI-TOF-TOF was done. Even though we do not provide a reason behind our observations, we guess dimethyl sulfoxide might have changed the charge distribution of peptides or proteins resulting in the peculiar observations we had.
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