Staining coomassie

Amido black is a commonly used stain, but it is not very sensitive. It is often used to visualize concentrated proteins or components that are readily accessible to dyes such as proteins that have been transferred from a gel to nitrocellulose paper. Two of the more sensitive and more frequently used stains are Coomassie Brilliant Blue (R250 and G250) and silver stains. Because these stains interact differently with a variety of protein molecules, optimization of the fixative and staining solutions is necessary. The Coomassie stains are approximately five times more sensitive than amido black and are appropriate for both agarose and polyacrylamide gels. The silver stain is approximately 100 times more sensitive than Coomassie and is typically used for polyacrylamide gels. 

Spray with a 0.03% solution of the Coomassie stain in 20% methanol Spray with a 5% solution in ethanol and heat at 100°C for 5 to 10 min Spray with a 10% solution of SbCl in chloroform heat at 110°C for 1 to 2 min... 

Fig. 8.4 Coomassie-stained SDS-polyacrylamide gel showing chloroplast transgenic lines expressing IFNa2b. Lanes 1 and 2 Total soluble protein (TSP) Lanes PH, 3 and 4 Total protein (TP).

Fig. 7.11. The rate of ubiquitination by the SCF - is dependent on the lysine-destruction motif spacing. (A) Sequences of the wild type and mutant / -catenin and kBa peptides, with the destruction motif and ubiquitinated lysine(s) highlighted. (B) Time courses of ubiquitination of the wild-type and mutant peptides, visualized by Coomassie staining. (C) The reaction yields plotted with error bars from four experiments. (D) The...

The minimal amount of applicable protein depends on the detection method. Mosdy detection by Coomassie staining is tenfold less sensitive than by silver staining, which drops down to 1 ng per band. 

The minimal amount of loaded protein depends on the detection method. As a rule of thumb, for Coomassie staining at least 1 pg of protein per band is needed. 

About 1 - 5 pi of the pre-mixed ready-to-use protein standards per lane are recommended in SDS-PAGE combined with Coomassie staining. 

Because the ability to dye one protein may differ from that of another in an electropherogram, an unknown protein mix should become stained by different methods. Some of the procedures can be done sequential in the same gel, e.g., additional Coomassie staining after silver staining. 

Staining can also be used for quantifying proteins in gels. The gel is scanned and the obtained picture is calculated by densito-metric software. The amount of protein is proportional to the peak area of the whole band. Since the reaction of a protein with a dye can vary in abroad range (row Bradford in Table 1.1 corresponds to Coomassie staining), caUbration of the intensity is only possible when the same or a very similar protein is used. 

The Coomassie staining solution is acidic enough to stop proteinase activity. 

Figure 8.8 SDS-PAGE of purification of (R)-ADH from L brevis (Riebel, 1997). Left Coomassie stain right silver stain.

CA stoichiometry of three copies of subunit E has been reported for the bovine brain-coated vesicle enzyme based on densitometry of Coomassie-stained SDS polyacrylamide gels (Xie, 1996). 

Scan the ECL-developed film, the Coomassie-stained nitrocellulose membrane, or a lane of the Coomassie-stained gel (in which standard proteins or the sample were separated) with a computing densitometer of sufficient resolution. 

When the images are perfectly aligned, the immunoreactive band can be distinguished in the Coomassie stained lane and its MW or pi determined by comparison with the appropriate lane. 

Ranganathan, V. and De, P. K. (1995) Western blot of proteins from Coomassie-stained polyacrylamide gels. Anal. Biochem. 234, 102-104. 

Ideally, the gel should have been stained with a Coomassie stain—preferably a colloidal Coomassie if detection sensitivity is an issue, e.g., (2,3), or one of the commercially available stains. This method is also compatible with Sypro stains (though an ultraviolet transilluminator will be required to visualize the protein bands or spots during excision). Standard silver stains are not compatible, but certain modified silver stains—e.g., (4), or one of the commercially available mass spectrometry compatible silver stains—can be used however, even these usually give inferior mass spectrometry data compared to Coomassie- or Sypro-stained gels. 

If external calibration of the spectrum is to be used, a spectrum of the calibration standards should be acquired immediately, using the same power setting. Figure 1 shows a typical MALDI spectrum obtained from the trypsin digestion of a weak colloidal Coomassie-stained ID gel band. 

Coomassie staining is only useful when high concentrations of chemokines are available (>1 pg per band). We have never used Coomassie staining for chemokines, because silver staining allows detection of less than 10 ng chemokine and therefore saves material. 

Fig 2. Expression of chemokines with and without N-terminal tags. Murine RANTES and human herpesvirus 8 vMIP-lB were expressed either as mature proteins (native) or with the addition of the residues MKKKWPR- to the N-terminus (K3) using the T7 based expression vector pET24d in either BL21(DE3) or BL21(DE3)pLysS cells. Each lane of this Coomassie stained SDS-polyacrylamide gel contains 0.04 OD600 units of the indicated strain following a 3-h induction. 

Fig. 4. Visualization of several purified RANTES proteins on a Coomassie stained SDS page Lanes 1 and 2, 6xHis-tagged RANTES expressed in the periplasmic space of E. coli Lane 3, Met-RANTES expressed in the cytoplasm of E. coli and purified from inclusion bodies Lane 4, eucaryotic expressed 6xHis-tagged RANTES.

From the Rf value(s) of the protein band(s) in your unknown sample, determine the molecular weights of these proteins by interpolation on your standard curve. Flow many bands did you observe in your unknown What wasAvere the molecular weight(s) If there was more than one band in your unknown, what can you say from the intensity of Coomassie staining of the bands about the relative abundance of the proteins in the sample ... 

Cut a notch in the upper corner of each half of the gel, next to lane 1 and lane 6. This will help you determine the orientation of the gel following Coomassie staining and transfer to the PVDF membrane. 

For the identification of proteins, preparative gels with a total protein load of 1 mg per gel were mn and proteins were visualized by Coomassie Blue R staining. Leflunomide binding proteins were analyzed using preparative SDS-PAGE. Coomassie-stained protein bands were identified by MS. 

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