Staining of proteins after electrophoresis

CBB R250 or G250 (0.1%) in methanol-water-glacial acetic acid (5 5 2), filtered before use, is the most frequently used stain. The gel is incubated in this solution for 1 h for SDS-containing gels the volume of the stain should be at least 10 times larger than that of the gel and destained by electrophoresis or by simple diffusion in 7.5% glacial acetic acid and 5% methanol in water (renewed from time to time to discard the unfixed stain). 

The various silver stains are more tedious but have a much higher detectability, particularly in the methods of Morissey (1981) and Marshall (1984) for black-and-white staining and of Sammons et 

Protein bands across the gel point to contamination. The presence of 2-ME may give two artificial bands corresponding to apparent molecular weights of 68000 and 54000 (Tasheva and Dessev, 1983 the role of 2-ME has been both confirmed and disputed by others). 

Protein streaking from top of the gel points to overloading or to protein precipitation (too little SDS, wrong ionic strength or pH). Streaking in the gel as may be apparent after silver-staining points to (bacterial) contamination of the solutions mixed. 

Precipitation in SDS systems is often due to the presence of K. A small amount of K will precipitate SDS, as will several other metal ions. 

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Figure 4.9. Staining of Proteins After Electrophoresis. Proteins subjected to electrophoresis on an SDS-polyacrylamide gel can be visualized by staining with Coomassie blue. [Courtesy of Kodak Scientific Imaging Systems.]...

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. 

Sinha, P. Poland, J. Schnolzer, M. Rabilloud, T. A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis. Proteomics (Germany) 2001, 1(7), 835-840. 

Disc gel electrophoresis yields excellent resolution and is the method of choice for analysis of proteins and nucleic acid fragments. Protein or nucleic acid bands containing as little as 1 or 2 ju,g can be detected by staining the gels after electrophoresis. 

K14. Koiw, E., and Gronwall, A., Staining of protein-bound carbohydrates after electrophoresis of serum on filter paper. Scand. J. Clin, iz Lab. Invest. 2, 244... 

L12. Loeffler, W., and Wunderly, C., The staining of proteins and lipoids after electrophoresis on filter paper. J. Clin. Pathol. 4, 282 (1953). 

Unlike ELISA, which can detect and quantitate host-cell proteins as a group, Western blots detect single protein impurities. Western blot analysis starts with an SDS-PAGE protocol but does not include a final colorimetric staining step. Instead after electrophoresis the proteins are electrotransferred (blotted) from the gel onto a thin membrane. Membranes made of nitrocellulose or PVDF are most often used. Once the transfer is complete, the membrane is incubated with a nonspecific protein solution to saturate and to... 

Figure 20-10 Comparison of immunofixation electrophoresis (IFE) and immunoelectrophoresis (lEP) for two patients with monoclonal gammopathies. A, Patient specimen with an IgG (kappa, k) monoclonal protein as identified by IFE.The arrow indicates the position of monoclonal protein.After electrophoresis, each track except SPE is reacted with its respective antiserum, then all tracks are stained to visualize the respective protein bands. (SPE Chemically fixed serum protein electrophoresis IgG, IgA, IgM, k, and A, indicate antiserum used on each track.) B, Same specimen as in A, with proteins identified by lERThe arrow indicates the position of monoclonal protein. Norma control (C) and patient sera (S) are alternated. After electrophoresis, antiserum is added to each trough as indicated by the labels ig (polyvalent Ig antiserum), IgG, IgA, IgM, K, and A The antisera react with separated proteins in the specimens to form precipitates in the shape of arcs.The IgG and k arcs are shorter and thicker than those in the normal control, showing the presence of the IgG (k) monoclonal protein.The concentrations of IgA, IgM, and A,-iight chains are also reduced. C, Patient specimen with an IgA (lambda, A,) monoclonal protein identified by IFE procedure as described in A. D, Same specimen as in C with proteins identified by lEP as described in B.The abnormal IgA and A,-arcs for the patient specimen indicate an elevated concentration of a monoclonal IgA (A.) protein. Ait separations were performed using the Beckman-Coulter Paragon system.

A photograph of one such gel, with protein bands made visible by Goomassie Blue staining is shown at the top of the figure ( 6 stains very poorly with this dye). An identical gel was treated with NH OH and GH COOH to cleave the crosslinks, then place on top of an SDS-polyacrylamide slab gel, and electrophoresis in the second dimension was carried out. After the gel was dried, 5h- labeled protein spots were visualized by autoradiography. The arrows indicate the direction of migration of proteins during electrophoresis. 

Ohsawa, K, and Ebata, N., 1983, Silver stain for detecting 10 femtogram quantities of protein after polyaciylamide gel electrophoresis. Anal. Biochem., 135 409 15. 

Figure 7.1 Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing agents, which disrupt disulfide linkages). The electric field is applied across the gel after the protein samples to be analysed are loaded into the gel wells. The rate of protein migration towards the anode is dependent upon protein size. After electrophoresis is complete, individual protein bands may be visualized by staining with a protein-binding dye (a). If one well is loaded with a mixture of proteins, each of known molecular mass, a standard curve relating distance migrated to molecular mass can be constructed (b). This allows estimation of the molecular mass of the purified protein...

After electrophoresis, protein bands are transferred onto a solid support. Many aspects of a transfer can affect antigen detection. Some of these factors are specific to the transfer method and choice of membrane, whereas others apply to the entire blotting procedure. For example, the transfer efficiency may be affected by the presence of SDS in the gel and whether the gel was stained prior to transfer. 

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... 

Amido black dye Two years later, Grassman and Hanning developed another organic dye to be used on filter paper after electrophoresis amido black stain. It has moderate sensitivity. Today, amido black dye is used for colorimetric determination of electroblotted proteins on PVDF (poly-vinylidene difluoride) and nitrocellulose membranes. 

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-... 

Detection of proteins on thin-layer plates, gel slabs, or membranes is often accomplished by staining with a dye,265-267 the most widely used being Coomassie brilliant blue.268 Various silver-containing stains may also be used. After separation of a protein mixture by electrophoresis and transfer to an inert membrane,... 

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