Staining gels

Progress of Ihe reaction should be monitored periodically during the 2 hr to determine when the starting material is consumed in order to avoid unnecessary heating, which leads to the formation of by-products. TLC can be used 50% ethyl acetate/hexane, silica gel stained with phosphomolybdic acid. 

Several types of imaging systems and associated software are commercially available for analyzing gels stained with just about any kind of stain.9(192 These instruments greatly simplify data acquisition and analysis and the archiving of gel patterns. 

Protein molar mass standards covalently labeled with dyes are valuable when the electrophoresis is followed by a Western blot or when electrophoresis has to be monitored. Furthermore, procedures of gel staining alter the geometry, and assignment of bands on blot to marker bands within the gel is sometimes difficult. 

Figure 5.1 Two-dimensional electrophoresis separates potato proteins based on their isoelectric points and molecular weights. Using a wide-scale pH gradient and large gels, 1000-2000 quantifiable proteins can be separated. The gel stained with SYPRO Rubi shows soluble tuber proteins of potato cultivar Sante.

Electrophoresis. SDS polyacrylamide gel electrophoresis (4-20% gradient) was performed using the method of Laemmli (20). Autoradiographic analysis was performed on gels stained with Coomassie Brilliant Blue (R-250), dried, and stored at -70 C with a sheet of X-ray film (Kodak X-OMAT RP) in direct contact with the gel. 

Sigma-Aldrich. Electrophoresis — Gel stains, Products for Life Science Research , 2000, pp. 825-829. www.sigma-aldrich.com. 

If an autoradiogram of a gel can be prepared, a permanent record of the experimental data is available. For biomolecules on a gel stained with Coomassie Blue (proteins) or ethidium bromide (nucleic acids), the best method for permanent storage is conventional photography. 

Prepare a table of relative mobilities of all bands in the gel. Compare the profile for isolated a-lactalbumin to that of standard a-lactalbumin. Is a-lactalbumin the predominant protein in your preparation from milk In whey Try to estimate the percentage of the isolated sample that is a-lactalbumin. Assume that all proteins on the gel stain to the same extent with the dye, even though this is probably not true. Approximately what percent of total whey proteins is a-lactalbumin Use the molecular weight standards to estimate the molecular weight of a-lactalbumin and other proteins. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Figure B3.1.2 Native discontinuous polyacrylamide gels activity stained for proteinases. (A) Gel stained with Coomassie brilliant blue for total protein. (B) Gel assayed for proteinase activity using casein as a substrate. Samples are enzyme extracts of hepatopancreas from four shrimp species. Lane 1, molecular weight markers Lane 2, Rcaliforniensis Lane 3 R vannamei Lane 4, Rpaulensis, Lane 5, P. schmitti.

Proteins in the middle two-thirds of the gel usually transfer to high-retention PVDF membranes with an average yield of 50% to 80%. The protein pattern on PVDF membranes stained with amido black or Coomassie blue should closely resemble the pattern on duplicate lanes of the gel stained with Coomassie blue. The staining intensity on the blot should be slightly higher than on the gel because the proteins are concentrated on the surface of the blot rather than distributed throughout the thickness of the gel. Proteins below the dye front usually will not be recovered on the membrane. Proteins in the top 20% of the gel are often incompletely transferred out of the gel. 

Fig. 1. Purified proteins imaged using (A) Agilent 2100 Bioanalyzer and (B) traditional sodium dodecyl sulfide-polyacrylamide gel electrophoresis. (A) Note that the lowest two bands and the uppermost band are internal standards for sizing and quantitation. (B) 3-15% polyacrylamide gel stained with GelCode Blue. In both panels, the predicted size of the protein is labeled at the top of each lane.

Figure 10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of products of limited proteolysis of the debranching enzyme with trypsin. The molecular weights shown of the various bands were determined by the methodology described previously (26). The ratio of debrancher to trypsin was 100 to 1. The incubation was conducted for 60 minutes at 25°. The gel stain was Coomassie Brilliant Blue and the absorbance was measured at 600 nm using a Gilford gel scanner with a 0-1 O.D. chart scale (36).

Fig. (16)l Liquid dectrofocused pattern (A) electrophorised in gels stained by protein reagent (B) and agar diffusion (C) of anti-lactose antibodies. 

Fig. 22a-f Photograph of starch gel electrophoretic patterns of avian egg whites to which Fe59 had been added before electrophoresis, a, c, and e are photographs of the gel stained for protein with Amido black, and b, d, and f are the corresponding autoradiograms which indicate the position of the conalbumin in the starch gel. (Clark, J. R., D. T. Osuga, and R. E. Feeney (1963) Comparison of avian egg white conalbumins. (J. Biol. Chem. 238, 3621 [1963])). 

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