Filipin staining of lipoproteins in polyacrylamide gels: sensitivity and photobleaching of the fluorophore and its use in a double staining method.
We have developed a double staining procedure in which polyacrylamide gels are first stained with filipin to identify lipoproteins, and then with Coomassie Brilliant Blue (CBB) to identify proteins. Filipin staining when performed at 37 degrees C is both more rapid and more sensitive than previously published procedures. After only 5 min, 20 ng of low density lipoprotein (LDL) unesterified cholesterol/mm3 of band volume could be detected, and after 12 h, sensitivity reached 0.8 ng/mm3. A semilogarithmic relationship was found between the amount of LDL unesterified cholesterol applied and filipin fluorescence. Although rapid photobleaching of the fluorophore occurred during UV transillumination of these gels, such photobleaching actually resulted in maximizing of the signal:noise ratio, resulting in better definition of bands. Treatment of gels with filipin had no deleterious effects on the subsequent staining with CBB. This dual staining procedure should prove useful for studies in which both lipoproteins and proteins in plasma need to be documented in the same gel.
Smejkal
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Cleveland Clinic Foundation Research Institute, Department of Cell Biology, OH 44195.
Hoff
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HF
D002784:Cholesterol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005372:Filipin; D005453:Fluorescence; D008074:Lipoproteins / Q000032:analysis*; D010777:Photochemistry; D012394:Rosaniline Dyes; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*
D008074:Lipoproteins; D012394:Rosaniline Dyes; D005372:Filipin; D002784:Cholesterol; C004692:coomassie Brilliant Blue