FILIPIN STAINING OF LIPOPROTEINS IN POLYACRYLAMIDE GELS - SENSITIVITYAND 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 sta ining when performed at 37 degrees C is both more rapid and more sensi tive than previously published procedures. After only 5 min, 20 ng of low density lipoprotein (LDL) unesterified cholesterol/mm(3) of band v olume could be detected, and after 12 h, sensitivity reached 0.8 ng/mm (3). A semilogarithmic relationship was found between the amount of LD L unesterified cholesterol applied and filipin fluorescence. Although rapid photobleaching of the fluorophore occurred during UV transillumi nation of these gels, such photobleaching actually resulted in maximiz ing of the signal:noise ratio, resulting in better definition of bands . Treatment of gels with filipin had no deleterious effects on the sub sequent staining with CBB. This dual staining procedure should prove u seful for studies in which both lipoproteins and proteins in plasma ne ed to be documented in the same gel.