A new approach for on-line enrichment in electrophoresis of dilute protein solutions

Journal of Biochemical and Biophysical Methods
Melinda RezeliS Hjertén

Abstract

A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing col...Continue Reading

References

Sep 23, 2000·Electrophoresis·D M OsbournC E Lunte
Aug 1, 1956·Biochimica Et Biophysica Acta·P FLODIN, D W KUPKE
Nov 1, 1956·Archives of Biochemistry and Biophysics·S HJERTENA TISELIUS
May 1, 1963·Journal of Chromatography·S HJERTEN

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Citations

Feb 19, 2008·Journal of Biochemical and Biophysical Methods·Maria HjerténStellan Hjertén

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