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We have shown that the fluorescent dye Nile red can be used for the staining of protein bands in sodium dodecyl sulfate (SDS)-polyacrylamide gels (3). Unlike the current methods using Coomassie blue or silver (5,7,9,10), Nile red staining does not require the fixation of protein in the gel. This allows the direct electroblotting and further sequencing and immunodetection of protein bands (1). Nile red was used previously for the fluorescent staining of hydrophobic structures such as lipid droplets (6). In the case of proteins separated in SDS gels, Nile red interacts presumably with the hydrophobic core of SDS-protein complexes (4,8). In the current staining method developed in our laboratory (2,3), the bands are visualized, with a detection limit of 20 ng of protein per band, after 5-min staining with Nile red. In this work, we have carried out a detailed study in order to reduce the background fluorescence of Nile red stained gels. The modified method presented in this report has a higher sensitivity and requires a shorter staining time than the original procedure. Protein electrophoresis was run on a Mini-Protean® II apparatus (Bio-Rad, Hercules, CA, USA). To prevent the formation of pure SDS micelles in the gel (3,4), the stacking and separating gels (8 × 6 × 0.075 cm) and the running buffer contained 0.05% SDS. Following electrophoresis, the gel was stained with different staining solutions (see below) containing different amounts of Nile red (Sigma Chemical, St. Louis, MO, USA). In all experiments, the required volume of a concentrated solution of Nile red (0.4 mg/mL) in dimethyl sulfoxide (DMSO) was added to 50 mL of deionized water or the indicated solutions previously placed in a polypropylene box (12 × 7.5 × 7 cm). The mixture was agitated gently for about 5 s, the gel was immersed immediately in the resulting solution, and the plastic box wrapped with aluminum foil was agitated on an orbital shaker (at about 150 rpm) for the times indicated. To obtain a homogeneous staining, care was taken to ensure that the gel was completely covered with the staining solution. Unless otherwise indicated, the staining was performed at room temperature. After staining, the gel was briefly rinsed with water (for about 10 s) and finally visualized using an ultraviolet (UV) transilluminator (302 nm) and Polaroid film (Cambridge, MA, USA) or a charged-coupled device (CCD) integrating camera and the Molecular Analyst® software of the Gel Doc 1000 system (BioRad). Generally, images were obtained with integration times ranging from 4–6 s. Longer integration times (8–12

Detection of Five Nanograms of Protein by Two-Minute Nile Red Staining of Unfixed SDS Gels