New polyacrylamide matrices for drift-free isoelectric focusing.
The major cause for pH gradient decay and cathodic drift during isoelectric focusing in polyacrylamide gels has been found to be electroendo-osmotic flow generated by fixed charges in the gel matrix. These changes have the following causes: (a) trace impurities of acrylic acid in the co-monomers; (b) covalent incorporation of catalysts (persulphate and riboflavin 5'-phosphate) as terminal groups in polyacrylamide chains; (c) hydrolysis of amide groups to acrylic acid in the gel layer underneath the cathodic filter paper strip. The result of these fixed negative charges in the matrix is a movement of counter-ions with hydration water towards the cathode (i.e. electroendo-osmosis) with concomitant drift of pH gradient and focused protein zones in the same direction. It is impossible to cure the cathodic drift by increasing the pH of the anolyte, or decreasing the pH of the catholyte, or both, as previously suggested. One way to reduce the cathodic drift efficiently is to incorporate covalently in the matrix tertiary or quaternary groups (3-dimethylaminopropylmethacrylamide) in stoichiometric amounts as compared with the negative charges. This 'balanced' polyacrylamide displays zero drift for at least 5000 V.h, which is considered to be an ample time for equilibrium separation of protein species in isoelectric focusing.
D055598:Chemical Phenomena; D002621:Chemistry; D005486:Flavin Mononucleotide; D005470:Fluorometry; D007525:Isoelectric Focusing / Q000379:methods*
D005486:Flavin Mononucleotide