Coomassie blue staining samples

Sodium dodecyl sulfate-PAGE (SDS-PAGE) was conducted in a 0.5-mm thick 15% horizontal slab gel (77). Samples were prepared in buffer with dithiothreitol and heated to 100°C for 5 min. The gel was prerun for 3 h at 10°C, pH 8.3, and 150 V, and then samples were run for 3 h at 250 V. Protein was again visualized with silver or Coomassie Blue stain. 

Hydrolysis and ion exchange amino acid analysis of an aliquot of the gel band provides a more accurate estimate of the amount of protein than can be obtained by comparing relative Coomassie Blue staining intensities. By detecting samples that contain too little protein or that are at too low density... 

With the exception of studies on bovine serum albumin (BSA) and human transferrin, all other digests were carried out on Coomassie Blue-stained gel bands that had been excised from SDS polyacrylamide gels and submitted in eppendorf tubes to the internal protein sequencing service of the HHMI Biopolymer Laboratory/W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University (5). The BSA and transferrin samples were subjected to SDS-PAGE in the Keck Facility and were otherwise prepared as described (5). Proteins were quantified by subjecting 10-15% aliquots of all gel slices to hydrolysis and ion exchange amino acid analysis (5). 

NH2-temiinus resulting in a mass of about 30 IdD and 3) an external peptide standard (450 pmol) that was provided dry in an eppendoiftube and that had the same amino acid composition as the unique tryptic peptide insert but whose sequence was randomized. In the case of the two protein samples, 70 pmol of each had been subjected to SDS PAGE and were supplied either as Coomassie Blue stained gel slices or as a section of amido black stained PVDF membrane. In the case of the PVDF samples, an oversize piece of PVDF was included so that a section could be used as a digest control, and in the case of the gel samples, a blank section of gel was included for the same purpose in a separate eppendorf tube. 

The conventional slab gel technique SDS-PAGE is an important technique for the routine identity and purity analysis of biotechnology products [36]. A typical SDS-PAGE analysis, run under reducing conditions with Coomassie blue staining, is shown in Fig. 3.21 A. The heavy and light chains of the purified antibody samples are clearly seen in all of the loaded samples. Molecular weight (MW) markers that encompass the bands of interest are also loaded onto the gel. No differences have been detected between cultures run in batch, fed-batch or perfusion modes. 

To determine the protein concentration and purity, check a sample of each fraction in a Bradford assay (or in a photometer at 280 nm absorption) and by SDS-PAGE followed by Coomassie blue staining. 

To initiate disassembly, lamin aggregates (about 420 ng) assembled in vitro from affinity-purified lamins isolated from either embryo or p- S]methionine-labeled Kc nuclei should be mixed with 6.3 p. of oocyte extract in a final volume of 158 /a1. Samples should be separated into supernatant and pellet fractions after 90 min of incubation at 23°C and analyzed by SDS-PAGE and Coomassie blue staining or fiuorography as appropriate we chose to fractionate 75- t.l samples of the reaction described above. 

We have achieved good separation of polyunsaturated fatty acid esters from saturated, monosaturated and disaturated fatty acid esters and this has been used as a means of identifying the presence of polyunsaturates, especially fish oil, in samples (Fig. 1.2). However, if there is a considerable amount of trienoic fatty acids in the mixture, as in egg lipids, the separation is not very pronounced (Shantha and Ackman, 1991a). Nakamura, Fukuda and Tanaka (1996) have used similar TLC separation of marine lipids and were able to quantitate the amount of pol)amsaturated fatty acids by using scanning densitometry followed by Coomassie blue staining. 

FIGURE 1 A typical blot overlay experiment to detect protein binding partners. Many proteins are present in the Coomassie blue-stained gel (A) however, I-zyxin recognizes predominantly a 23-kDa protein (the cysteine-rich protein) from this complex protein sample (B). The purity of I-zyxin used in this assay is shown in C (750,000 cpm, exposed for 10 min). 

Fig. 1. Affinity purification of phosphorylated and nonphosphorylated Flag-ASAPl from transfected HEK293T cells. HEK293T cells transfected with the indicated constructs are lysed and incubated with anti-Flag affinity matrix. Following elution with 3xFlag peptide, purity of ASAPl and its phosphorylation status are analyzed by Coomassie blue staining (A) 10 fi of purified sample per lane and Western blotting with anti-phosphotyrosine (pY) or anti-ASAPl antibodies (B) 5 pi of purified sample per lane.

Fig. 2. SDS-PAGE analysis of [ C]SAM-treated ACC synthase preparations. A sample of ACC synthase preparation was incubated at 30 C for 6 h with S-adenosyl-L-[3,4- C]methionine and the resulting proteins were analyzed either before (lanes A and C) or after (lanes B and D) purification by the immunoaffinity gel against ACC synthase [4]. At least two of the bands eluted from the immunoaffinity gel (lane B) are the antibody IgG proteins. Lanes A and B are Coomassie blue stain, and lanes C and D are the fluorograms of lanes A and B respectively. The 50 kD arrow indicates the position of ACC synthase protein. From Satoh and Yang [27]...

FIGURE 5. Polypeptide analysis of Chl-proteins isolated on a sucrose gradient. 1, spinach thylakoids 2, Prochloron Chi -b and Chi a-b 3, Prochloron CPI 4, protein standards. Samples were boiled with SDS and Coomassie Blue stained. 

Figure 5. Densitiometric scans of electrophoretograms of hepatic microsomes from rainbow trout pretreated with polychlorinated biphenyl congeners (A), control microsomes, 90 fig protein/gel (B), 2,2, 4,4 -tetrachlorobiphenyl-induced microsomes, 90 fig protein/gel (C), 3,3, 4,4 -tetrachlorobiphenyl-induced microsomes, 90 fig protein/gel (D), Aroclor 1242-induced microsomes, 90 fig protein/ gel. The slab gels were stained with Coomassie Blue-250 and individual sample tracts were cut out and scanned at 550 nm. The vertical broken line is at 57,000...

Check transfer efficiency by staining the gel after transfer, or by staining a second blot with a total protein stain, such as coomassie blue or ponceau red. Alternatively, use commercially available prestained protein standards that are run along the samples of interest and that are visible during both the separation electrophoresis and on the membrane after transfer... 

Since ammonium sulfate fractionation will also cause precipitation of other proteins, antibody concentrations obtained from absorbance measurements at 280 nm are only estimates. Alternatively, a sample of the dialyzed solution can be resolved on a SDS-polyacrylamide gel alongside a series of known concentrations of IgG. Staining the gel with Coomassie blue can then be used to estimate the amount of immunoglobulin obtained and can also give an estimate of purity. 

FIGURE 3-19 Electrophoresis, (a) Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field, (b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins blit not to the gel itself. Each band on the gel represents a different pro-... 

In Experiment 4, your sample of a-lactalbumin extracted from bovine milk was subjected along with other proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After staining with the dye Coomassie Blue, deeply colored bands appeared on the gel wherever there was a protein. You suspected that some of the blue bands on the gel were due to a-lactalbumin. If molecular weight standards were included on the slab gel, you were able to estimate the molecular weight for a-lactalbumin and other proteins. SDS-PAGE is indeed a very effective analytical tool to achieve fractionation of protein mixtures, to analyze purity, and to estimate molecular weight, but it provides no experimental data to prove the identity... 

Fig. 1. SDS-PAGE minigel depicting purified IgG preparations derived from human scrum (lane 2), mouse ascitic fluid (lanes 3 and 4), rabbit serum (lane 5), and sheep serum (lane 6). Samples are electrophoresed under reducing conditions and stained with Coomassie blue. All IgGs consist predominantly of two bands comprising heavy (50,000 Mr) and light (22,000 Mr) chains with no major contaminating proteins. Molecular weight markers are shown in lanes 1 and 7 and their molecular weights given on the left.

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.

Adjust sample concentration to 0.5 to 10 mg/ml for Coomassie brilliant blue staining or 0.05 to 1 mg/ml for silver staining. 

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