Staining of gels

Separate DNA fragments on nondenaturing (for ds DNA) or denaturing (for is DNA) polyacrylamide gel. A small amount of labeled markers are included in a separate track to assess the efficiency of transfer. Staining of gel is generally only useful for ds DNA at about > 25 ng/band. 

Fig. 2 (a) Silver stain of gel electrophoresis of pure recombinant rat PAP (size in kDa). Lane 1 native PAGE. Lane 2. SDS-PAGE. (b) Immunodetection (size in kDa) of purified recombinant rat PAP by Western-blotting. Lane / native PAGE. Lane 2. SDS-PAGE. Immunodetection with rabbit polyclonal antibody against an N-terminal peptide of rat PAP (in house antibody) [3]... 

The fluorescent SYPRO stains are praised as a breakthrough in the protein staining of gels. 

Using the photochemical flux of OJ for activity staining of gels, one interfering reaction is often overlooked. In the presence of hydrogen peroxide, catalase yields also achromatic bands. Owing to the reactivity of catalase (Eq. (4)) this phenomenon must be seen in the local rise of the pO ... 

C. STAINING OF GELS WITH COOMASSIE BRILLIANT BLUE... 

The purity of the AE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pi lEF 3-9 PhastSystem gels were used. 

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.

Stain the gel with silver using standard procedures. Careful examination of the gel should reveal bands that appear in sample 2 and not sample 1, which can be assumed to be captured specifically by the biotin-conjugate. Furthermore, if these same bands are not present, or are lower in intensity in sample 3, they can be assumed to be specifically captured in sample 2, but lost in sample 3 due to occupation of the binding-site by the free compound during the pre-incubation step. 

Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850—858. 

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). 

Direct staining of proteins (e. g., after electrophoretic separation in polyacrylamide gels) can be achieved by treatment with dyes like Coomassie Brilliant Blue R-250 [146] (Fig. 7), which binds positively charged proteins in an acidic fixation buffer, allowing detection down to 0.1 pg of protein. 

Using an acrylamide-gel as support, Coombe- 0 has demonstrated the quantitative assay of neomycin with a recovery of 96% in the presence of bacitracin and polymixin. In this case quantitation was achieved by densitometry after staining the gel with naphthalene black. 

Despite some refinements in the methods, the basic principles and protocols of gel electrophoresis have not changed appreciably since their introduction. Proteins are introduced into a gel matrix and separated by the combined effects of an electrical field, buffer ions, and the gel itself, which acts as a protein sieve. At the completion of the electrophoresis run, separated proteins in the gel are stained to make them visible, then analyzed qualitatively or quantitatively. The topic has been covered in numerous texts, methods articles, and reviews.1-11 In addition, apparatus and reagents for analytical and preparative gel electrophoresis are available from several suppliers. 

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