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Fast and sensitive protein staining with colloidal acid Violet 17 following isoelectric focusing in carrier ampholyte generated and immobilized pH gradients
Author(s) -
Patestos Nikos P.,
Fauth Markus,
Radola Bertold J.
Publication year - 1988
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.1150090908
Subject(s) - staining , chemistry , chromatography , immobilized ph gradient , isoelectric focusing , isoelectric point , phosphoric acid , methylene blue , absorbance , biochemistry , organic chemistry , photocatalysis , enzyme , catalysis , medicine , pathology
A new method is described for fast and sensitive staining of proteins following isoelectric focusing in carrier ampholyte and immobilized pH gradient polyacrylamide gels. After fixation with trichloroacetic acid the gels are stained for 5–10 mm with 0.1–0.2 % colloidal Serva Violet 17 (generic name: Acid Violet 17; Color Index No. 42 650) in 10 % w/v phosphoric acid. After staining for only 0.5–3 min, major zones, corresponding to 100–500 ng protein, are visible without destaining on a weak background. Detection of minor components requires destaining with 3 % w/v phosphoric acid for 5–80 min depending on gel thickness (120–500 μm) and type of support (fabric reinforced versus gels backed to a polyester film). For selected pH marker proteins (bovine serum albumin, carbonic anhydrase, horse myoglobin) a staining sensitivity of 1–2 ng/mm 2 protein is found. Dye elution from stained fabric reinforced gels with 50 % v/v dioxane‐water, followed by absorbance measurements results in a linear relationship over a range of 1–100 μg marker proteins. Staining colloidal Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges. Staining with the colloidal dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazadous.