Fast and sensitive protein staining with colloidal acid violet 17 following isoelectric focusing in carrier ampholyte generated and immobilized pH gradients.
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 min 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 microns) 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/mm2 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 micrograms marker proteins. Staining with collodial 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 collodial dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazardous.
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Institut für Lebensmitteltechnologie und Analytische Chemie, Technische Universität München, Federal Republic of Germany.
Fauth
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M
Radola
B J
BJ
D003102:Colloids; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D012394:Rosaniline Dyes; D013194:Staining and Labeling; D013329:Structure-Activity Relationship
D003102:Colloids; D011506:Proteins; D012394:Rosaniline Dyes; C031693:violet BNP