Coomassie blue

Figure 4-4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein standards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

Figure 4-5. Two-dimensional lEF-SDS-PAGE.The gel was stained with Coomassie blue. A crude bacterial extract was first subjected to isoelectric focusing (lEF) in a pH 3-10 gradient. The lEF gel was then placed horizontally on the top of an SDS gel, and the proteins then further resolved by SDS-PAGE. Notice the greatly improved resolution of distinct polypeptides relative to ordinary SDS-PAGE gel (Figure 4-4).

Figure 52-3. Diagrammatic representation of the major proteins of the membrane of the human red blood cell separated by SDS-PAGE. The bands detected by staining with Coomassie blue are shown in the two left-hand channels, and the glycoproteins detected by staining with periodic acid-Schiff (PAS) reagent are shown in the right-hand channel. (Reproduced, with permission, from Beck WS, Tepper Ri Hemolytic anemias iii membrane disorders, in Hematology, 5th ed. Beck WS [editor]. The MiT Press, 1991.)...

A) Proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. Lane 1, 4 )ig purified PGl Lane 2, 2 pg purified PG2 Lane 3,2 pg purified subunit. 

Fig. 13.2. The peptide components of H-gal-GP and TSBP visualized by Coomassie Blue staining of non-reducing (lanes 1 and 3) and reducing (lanes 2 and 4) SDS-PAGE gels.

Fig. 3.2 Rapeseeds were germinated from 12to 168 h in airlift tank. The total soluble proteins were extracted and separated by 15% SDS-PAGE. The gel was stained with Coomassie blue. Between 36 and 60 h, the degradation of storage proteins and the de novo synthesis of Rubisco is clearly visible.

Fig. 3.7 Transgenic rapeseeds were sprouted in an airlift tank with (lane 1) and without (lane 2) of streptomycin at 100 mg L-1. Total proteins were extracted, separated by SDS-PAGE and stained with Coomassie blue. The synthesis of Rubisco large and small subunits was inhibited as clearly shown in lane 2.

Bradford reagent contains the dye Coomassie blue G-250 in an acidic solution. The dye binds to protein, yielding a blue colour that absorbs maximally at 595 nm Copper-containing reagent that, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm Essentially involves initial precipitation of protein out of solution by addition of trichloroacetic acid. The protein precipitate is redissolved in NaOH and the Lowry method of protein determination is then performed Interaction of silver with protein - very sensitive method... 

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product). 

After completion of electrophoresis, the gel was stained with Coomassie blue. To prepare the staining solution, 40 mg Coomassie blue R-250 was dissolved in 25 ml isopropanol and 10 ml glacial acetic acid. The solution was filtered and the volume was increased to 100 ml with distilled water. The gel was placed in staining solution for 1 h, followed by washing with 10% acetic acid/2.5% isopropanol. 

Densitometric scans of Coomassie Blue stained electrophore-tograms were obtained at 550nm, using an ISC0 gel scanner accessory in combination with an ISC0 Type 6 optical unit and Type UA-5 absorbance monitor. 

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...

Size-based analysis by CE provides similar information and comparable limits of detection to analysis by SDS-PAGE with Coomassie blue staining.120 129 The performance of both electrophoretic techniques for the analysis of polypeptides is far superior to size exclusion chromatography. Figure 9.7 shows the separation of SDS-complexed recombinant protein standards by CE. 

Protein concentration was determined using the Bradford assay at 595 nm. 100 pL of the sample were introduced into a cuvette containing 5 mL of Bradford solution (100 mg of Coomassie blue, 50 mL of ethanol and 100 mL of 85 % phosphoric acid dissolved in 850 mL of H2O). The solutions were incubated for 5 min at room temperature. The absorbance was measured at 595 nm. The protein concentration in the sample was determined using a calibration curve plotted with serum albumin (1 mg mL ) as a standard.)... 

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... 

For visualisation of proteins after separation on gel, one could use different stains such as Coomassie blue stain or more sensitive silver staining. The Coomassie blue staining is relatively less sensitive than silver staining, but is highly convenient to use. 

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. 

Two-dimensional electrophoresis [86] is a well established technique for the separation of intact proteins. In the first dimension the proteins are separated based on their isolectric point while the second dimension separates them based on their size. The presence on the gel of the proteins is revealed by Coomassie blue or silver staining. Under favorable conditions several thousand spots can be differentiated. The gel is digitized and computer-assisted analysis of the protein spot is performed. The spots of interest are excised either manually or automatically and then digested with trypsin. Trypsin cleaves proteins at the C-terminal side of lysine and arginine. In general one spot represents one protein and the peptides are analyzed by MALDI-TOF to obtain a peptide mass fingerprint. A peptide mass fingerprint involves the determination of the masses of all pep-... 

PAGE was carried out according to the modified method of Reisfield et al. (10), using Fuchsin Red as a marker. Gels were run under cathodic conditions and then stained with Coomassie Blue. 

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. 

Protein content of various fractions was determined with Coomassie Blue by the method of Bradford (46) as refined by Spector (47). 

Protein concentration was determined by use of Coomassie Blue reagent (11). 

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