Models of protein modification in Tris-glycine and neutral pH Bis-Tris gels during electrophoresis: effect of gel pH.
The pH of conventional Tris-glycine SDS-PAGE gels during a run is determined to be 9.5, in contrast to Bis-Tris-Mes gels where the pH is 7.2. Concentrations of free acrylamide are determined to be less than 10mM in commercial gels of both types, and it is found that of the major components in these gels, only glycine and protein amine or sulfhydryl functions are likely to react with residual acrylamide during the time frame of typical separations. The addition of acrylamide to sulfhydryl groups on proteins is modeled using glutathione and cysteine at acrylamide concentrations found in the commercial gels. Rate constants are determined for these reactions as well as for reaction with glycine at the pH that proteins will encounter in these gel types. The half-life for glutathione sulfhydryl at 10mM acrylamide and pH 7.2 is more than 4h at room temperature. Rates are significantly lower in Bis-Tris-Mes gels than in Tris-glycine gels, reducing the risk of adventitious protein modification. Commercial Bis-Tris-Mes gels provide a sample reduction buffer at pH 8.5 versus the conventional pH 6.8 of Tris-glycine gels. It is shown that significantly less protein degradation occurs during sample preparation at the higher pH used with Bis-Tris gels.
Hachmann
John P
JP
Invitrogen Corporation, Carlsbad, CA 92008, USA.
Amshey
Joseph W
JW
D020106:Acrylamide / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D002021:Buffers; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005978:Glutathione / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014325:Tromethamine / Q000031:analogs & derivatives / Q000737:chemistry
D000588:Amines; D002021:Buffers; D010455:Peptides; D011506:Proteins; D013438:Sulfhydryl Compounds; D014325:Tromethamine; D020106:Acrylamide; D005978:Glutathione; D003545:Cysteine; C026272:Bistris; D005998:Glycine