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High resolution two‐dimensional polyacrylamide gel electrophoresis of basic myeloid polypeptides: Use of immobilized pH gradients in the first dimension
Author(s) -
Strahler John R.,
Hanash Samir M.,
Somerlot Luke,
Weser Johann,
Postel Wilhelm,
Görg Angelika
Publication year - 1987
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150080402
Subject(s) - isoelectric focusing , chromatography , immobilized ph gradient , chemistry , urea , electrophoresis , polyacrylamide gel electrophoresis , polyacrylamide , gel electrophoresis , resolution (logic) , silver stain , two dimensional gel electrophoresis , sodium dodecyl sulfate , analytical chemistry (journal) , biochemistry , microbiology and biotechnology , polymer chemistry , artificial intelligence , computer science , gene , proteomics , biology , enzyme
A two‐dimensional polyacrylamide gel electrophoresis approach for the separation of urea/detergent solubilized basic proteins is presented. An immobilized pH gradient (IPG) gel, pH range 7–10, containing 8 M urea, 2% Nonidet P‐40 and 10 mM dithioerythritol was used for the first‐dimensional isoelectric focusing separation. Approximately 400 to 500 basic polypeptides from myeloblasts or neutrophils were visualized with silver staining. The IPG gel in some cases included carrier ampholytes in the same pH range. The carrier ampholyte‐modified (“hybrid”) IPG gel exhibited improved resolution of some polypeptides, but at the expense of reproducibility in the focusing of other polypeptides. Transfer of sample into the IPG gel was facilitated by a combination of low initial voltage, presence of carrier ampholytes in the sample and application of the sample to the surface of the gel within a containment collar. 6 M urea was required in the SDS equilibration solution for the efficient transfer and high resolution separation of polypeptides in the second‐dimensional sodium dodecyl sulfate‐gel. Absolute spot position was highly reproducible among gels prepared simultaneously. Standard deviations of ± 0.84 mm for the focusing and ± 1.25 mm for the molecular weight dimensions of 70 randomly selected spots were obtained. Integrated spot intensities ranged from 0.17 to 80 O.D. × mm 2 with a median of 1.5 O.D. × mm 2 .