Coomassie blue staining protein detection

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. 

Fig. 1. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of expression and purification of recombinant protein. Ten-microliter aliquots were withdrawn at each step of the purification and loaded on a 12% SDS-PAGE gel in a Mini Protean III cell gel electrophoresis unit (Bio-Rad). The detection was performed with Coomassie blue staining. MW, low range (14-98 kDa) molecular weight marker (Bio-Rad).

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... 

Protein is invisible in the gels and must be stained for detection. The most commonly used visualization techniques are silver and Coomassie blue stains. While silver is more sensitive, the intensity of silver stains is affected by the proteins and is not linear with the concentration of protein, as is Coomassie blue staining. If the intention is to quantify the relative amounts of each protein band, Coomassie blue staining should be used. 

Silver staining of proteins after their separation by electrophoresis is based on binding of silver ions to sulfhydryl and carboxyl groups of proteins. The proteins are detected as black precipitate of silver. The sensitivity of this method is 20-100 times more sensitive than Coomassie blue staining. 

Of particular interest are the phosphorylated species of 10 and 13 kDa. Under the conditions employed the 10 kDa protein was strongly labelled in the light whereas the 13 kDa band was weakly labelled, but in the dark neither species was phosphorylated to a detectable level. Both these labelled bands were coincident with Coomassie blue staining bands (Fig. 1, lane 1)... 

Figure 1 shows the proteins contained in various subnuclear fractions. Histones were not detectable in the isolated nuclear matrix by Coomassie blue staining. But these known acceptor proteins for ADP-ribose were found in the ammonium sulfate extract. Autoradiography revealed that great amounts of the self-modified poly(ADP-ribose) synthetase - in accordance with the results of Table 1 - were released by DNase, RNase digestion of the isolated nuclei (not shown). 

Transfer the proteins after SDS-PAGE to a polyvinylidene fluoride (PVDF) membrane and detect the proteins by specific antibodies at 1 2,000 dilutions using Western blot procedures, or by Coomassie Blue staining. Alternatively the proteins can be identified by mass spectrometry as will be described in the following paragraph. 

We have used the Protein A280 function of a NanoDrop 1000 for monitoring protein elution peaks which consumes only 2 pi elute each time but when elute is too diluted for a NanoDrop detection, SDS-PAGE with Coomassie Blue staining or Western blotting detection can be used. 

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. 2. SDS PAGE analysis of rCRALBP. Approximately 10 pg of soluble protein from crude lysates containing rCRALBP and 1-2 pg of puriHed fusion and nonfusion rCRALBP was analyzed according to Laemmli (27) on a 12% gel. Detection is by Coomassie blue staining.

Fig. 1. Electrophoretic analysis of P. cruentum thylakoid membranes (A) unstained gel scanned at 675 nm to detect Chi and (B) Coomassie blue stained gel scanned at 590 nm to detect protein. Peaks I, III, IV are the Chl-protein complexes. The stained gel is shown at the bottom.

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 variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

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