A new approach for on-line enrichment in electrophoresis of dilute protein solutions.
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 colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.
Rezeli
Melinda
M
Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Ifjúság útja 6., H-7624 Pécs, Hungary.
Kilár
Ferenc
F
Hjertén
Stellan
S
D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods*; D007201:Indicator Dilution Techniques; D015394:Molecular Structure; D009862:Online Systems / Q000295:instrumentation*; D011506:Proteins / Q000032:analysis*; D012996:Solutions
D011506:Proteins; D012996:Solutions