ISSN:
0173-0835
Keywords:
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Ultrathin-layer isoelectric focusing in polyacrylamide gels on silanized supports provided the means for developing a miniature system combining short focusing time with high resolution and operational simplicity. The essence of miniature ultrathin-layer isoelectric focusing is the combined use of short separation distances (1-3 cm) and high field strengths (400-700 V/ cm), which can be applied in standard equipment simply by reducing the thickness of the gel to 20-50 um for improved heat dissipation. By the criterion of coalescence of proteins migrating from different positions, a steady state is reached in pH 4-9 servalyt T carrier ampholytes on 1 cm gels after 2 min (15 Vh) and on 3 cm gels after 10 min total focusing time (110-130 Vh). On 3 cm gels the resolution is 0.03-0.035 pI in wide-range carrier ampholytes, and 0.01-0.02 pI in narrow-range (0.3 pH units) carrier ampholytes isolated by preparative isoelectric focusing of pH 4-9 Servalyt T in Bio-Gel P-60 layers. Miniature ultrathin-layer isoelectric focusing can easily be adapted to the analysis of multiple samples. When the volume is limited to 0.2-0.4 μl, up to four samples per cm can be applied directly on the gel surface. At this loading, the gel volume per focused sample amounts to only 0.5-5 μl. The gel volume per sample is reduced by a factor of 10-200, relative to the 10-12 cm separation distance in 50-100 μm gel layers. This results in a considerable saving of carrier ampholytes and reagents for gel preparation. For serial dilutions of pH marker proteins, the sensitivity of protein detection after staining with Serva Blue G or Serva Violet 49 is 10-15 ng protein in the miniature system. Miniature ultrathin-layer isoelectric focusing should prove to be an attractive method for routine applications in clinical, industrial and research laboratories in those cases when only small amounts of material are available or when rapid results are required. The method is particularly suitable for studies of genetic polymorphisms of proteins and enzymes.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150020309
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