Biochemistry and Biotechnology
Wiley InterScience Backfile Collection 1832-2000
Chemistry and Pharmacology
Ultrathin-layer isoelectric focusing in 50-100 μm polyacrylamide gels covalently bound to glass plates or polyester films, pretreated with methacryloxypropyl-trimethoxy-silane, is described. The gels adhere firmly to the silanized supports during all steps of operation and visualization. Ultrathin gels are prepared by the new and simple “flap technique”. Artificial mixtures of marker proteins and crude fungal enzymes were focused on 5-25 cm separation distances. In 11 cm gels containing pH 4-9 “Servalyt T” carrier ampholytes, 2-3 zones per mm can be resolved, with a zone width of 70-150 μm and differences in pI values of 0.023-0.033 pH. The gel volume per focused sample is 15-120 μl and up to 100 samples can be analyzed on 12.5 × 26 cm gels. Polyacrylamide gels of intermediate and high porosity, e. g. 5 % T, 3 % C and 3 % T, 20 % C, are compared. Small marker proteins attain equilibrium in both gels after focusing for 1300-2000 V x h at a final field strength of 100 V/cm, whereas thyroglobulin (690 000) and ferritin (465 000), applied at the anode and cathode, coalesce only in high-porosity gels. pH Gradients were determined, with the aid of a glass electrode, directly on the gel surface for wide range carrier ampholytes “Servalyt T”, “Ampholine”, and “Pharmalyte” after focusing for 2000-5000 V × h. Identical focusing patterns are found for some marker proteins and crude fungal enzymes in different carrier ampholytes. The effect of purification of commercial carrier ampholytes by ultrafiltration and charcoal treatment, and of “forced aging” at 80°C, on isoelectric focusing of marker proteins was studied. The molecular size distribution of different carrier ampholytes was assessed by thin-layer gel chromatography on Bio-Gel P-10. Protein staining with Serva Blue G, Serva Blue R and Serva Violet 49 is described. The sensitivity of protein detection is 50-100 ng/mm2. Fixation, staining and destaining can be completed within 10-15 min in 50 μm gels. Ultrathin-layer isoelectric focusing on silanized glass plates or polyester films recommends itself as an excellent analytical technique for research and routine applications. The method combines high resolution, speed, versatility and reagent economy with simplicity of operation.
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