Sunset Yellow: A Food Color for Protein Staining with SDS-PAGE

Abstract

This study shows that Sunset Yellow FCF is a fast-reacting dye for food proteins and may be used as a fast staining agent for all types of resolved proteins with PAGE, which is important because Coomassie Brilliant Blue R 250 and Amido Black 10 B take at least 24 hr for visualization of proteins bands with PAGE. Sunset Yellow FCF is highly sensitive, rapid, lasting, and provides reproducible results with a variety of proteins. It produces sharp, nondiffuseable, clear, distinct, and stable bands on gel. It requires just 1 hr for staining and destaining to visualize the known proteins separated on the acrylamide gels. intestinal anaerobes (5). The dye may be purified by a chemometric method (6) and, even in its crude form, is equally effective for protein staining on acrylamide and for use after blotting (7). Apart from Coomassie Brilliant Blue, the other standard protocols of dyes include silver and gold staining (8–10) and Amido Black 10 B (11); iodine is less commonly used (12). Although the spectrophotometric determination and estimation of Sunset Yellow FCF at 485 nm is common, capillary electrophoresis has recently been used as a sophisticated technique to estimate colors in foods, including this dye in beverages (13,14). The nanomaterials in foods, such as micronutrients and synthetic colors like Sunset Yellow FCF, may be analyzed by the above separation technique. The dyebinding assays are widely used in protein analysis. Our data show that Sunset Yellow FCF is a sensitive, reliable, and competent dye for binding proteins to make them visible with PAGE. MATERIALS AND METHODS Chemicals N,N′-methylene-bis-acrylamide was purchased from Scharlau, while Tris (hydroxymethyl) aminomethane was obtained from Research Organics. Sodium dodecyl sulfate (SDS), acrylamide, ammonium peroxodisulfate (APS), glycine, Coomassie Brilliant Blue, bovine serum albumin (BSA), N,N,N′,N′-tetramethylenediamine (TEMED), bromophenol blue, and trypsin were supplied from Merck, while nisin was purchased from Suzhou Hengliang Co. Ltd., Suzhou, China. The 2-mercaptoethanol and HCl were supplied from Riedel-deHaen, Seeize, Germany. Sunset Yellow FCF No. 6 was a gift from coauthor Asad Sayeed (National Foods Ltd., Karachi, Pakistan). Glycerol and takadiastase from Aspergillus oryzae were purchased from Fluka, Steinhein, Denmark. Preparation of Slab Gel The following solutions were first prepared. For solution A, acrylamide (30 g) and bis-acrylamide (0.80 g) were dissolved in 100 mL of double-distilled, deionized water and filtered through Whatman No. 1 filter paper. For solution B, Tris-HCl buffer (1.5M) was prepared by mixing 18.02 g of Tris dissolved in 80 mL of double-distilled, deionized water, and the pH was adjusted to 8.8 by using 1M HCl; the volume was made up to 100 mL. For solution C, Tris (12.114 g) was added to 80 mL of doubledistilled, deionized water; the pH was adjusted to 6.8 with 1M HCl; and the volume was made up to 100 mL with double-distilled, deionized water. For solution D, SDS (10 g) was dissolved in double-distilled, deionized water, and the volume was made up to 100 mL. For solution E, APS (1 g) was dissolved in double-distilled, deionized water, and the volume was made up to 10 mL, while TEMED was used as supplied. The 12.5% slab gel (thickness 0.75 mm) was prepared by using 2.083 mL of solution A, 0.666 mL of solution B, 0.500 mL of solution D, and 0.250 mL of solution E. The mixture was degassed, and 1.66 μL of TEMED was added before use. The gel was left for 15 min to settle. Staking Gel Solutions A, C, D, and E were added in the ratio of 0.416:0.830:0.333:0.166, respectively. The mixture was immediately degassed, and 1.66 μL of TEMED was added before the stacking gel was poured. The wells were prewashed with reservoir buffer after the gel had settled. For the reservoir buffer, Tris (0.9 g), glycine (3.6 g), RESEARCH