Silver-stained SDS-PAGE

Figure 14 6 Silver-stained SDS-PAGE gel of PatA binding proteins. Lane 1, sample 1 nonspecific proteins captured by the streptavidin-agarose resin Lane 2, sample 2 proteins affinity captured by the presence of B-Pat A Lane 3, sample 3 affinity capture of target proteins was blocked by prior addition of free PatA before incubation with B-PatA. The two arrows point to two proteins specifically detected in sample 2 versus sample 1, which were also lost due to competition in sample 3, with apparent molecular weights of 38 and 48 kDa.

Careful sample handling is important when sensitive detection methods are employed. Silver-stained SDS-PAGE gels sometimes show artifact bands in the 50- to 70-kDa molecular mass region and irregular but distinctive vertical streaking parallel to the direction of migration. The appearance of these artifacts has been... 

Figure 3 Silver stained SDS-PAGE gel of iPLA2-VI purification. Lane 1, molecular mass markers lane 2,20 pi of pool from octyl-Sepharose column lane 3,20 pi of fraction 28 from the Calmodulin-agarose column lane 4,20 pi of fraction 29 from the Calmodulin-agarose column and lane 5,20 pi of fraction 30 from the Calmodulin-agarose column. (From Ref. 41.)...

Second, methods for the characterization of complex antisera are difficult. Antisera to E. coli protein mixtures have been developed with impressive spectra of reactivity using conventional immunization methods (6,22-23). An exact assessment of the spectrum of antibody reactivity is often limited, however, by the resolution of the analytical methods used. Counter immunoelectrophoresis is limited by the relatively low sensitivity of detection and resolution for complex mixtures of reacting species. One dimensional silver stained SDS-PAGE and immunoblotting provides sensitive detection limits but lacks resolution. Therefore, methods which have a high degree of resolution and sensitivity are required to best compare potential improvements in the production of antibodies to minor components in the mixture. 

The third group underwent a modification of the cascade immunization procedure (24). After a primary injection of ECP in CFA and a subsequent injection in ICFA a serum sample was taken seven days later and the IgG fraction used to prepare an affinity column. The entire ECP mixture was passed over the column and fractions which were depleted of one or more ECPs (as determined by silver stain SDS-PAGE and compared to the starting preparation) were used for the subsequent immunization injection. This procedure of ECP adsorption was repeated with subsequent antisera obtained on days 28 and 42 and the resulting depleted ECP fractions used for injections on either days 35 or 49 and 62, respectively (Figure 3). Thereafter the rabbits received injections as described in the normal immunization procedure. 

It was clear from this study that affinity chromatography with early antibodies (after careful analysis and pooling of the column flowthrough by a very sensitive method such as silver stain SDS-PAGE) can be used to alter the distribution of the reference immpurity protein mixture toward those components which were in low concentration or poor immunnogens. 

Figure 3. Selection of ECP subpopulations forjprogressive iterations of the cascade procedure by silver stained SDS-PAGE. Lane 2 in each panel shows the entire ECP mixture used as the column load and lane 3 shows the column flowthrough fraction used for the next injection. Panel A demonstrates the affinity chromatography performed with day 14 antisera, Panel B with day 28 antisera and Panel C with day 42 antisera. The arrow shows ECPs depleted by the early antibodies. The progression of the immune response is clearly apparent although it is clear not all of these proteins are equally immunogenic. A 50 Kd protein has saturated its respective antibody and begun to flow through the column (Panel C, lane 4). Reproduced with permission from Ref. 24. Copyright 1989 The Humana Press Inc.

Figure 5.1-7. SDS-CGE separations of nonreduced and reduced preparations of a therapeutic recombinant MAb. Insets show silver-stained SDS-PAGE traces of the same sample preparations (M monomer, A aggregate). (A) UV detection of unlabeled samples at 220nm. (B) LIF detection of labeled samples at 488-nm excitation and 560-nm emission. (Reprinted from Ref. 93, with permission.)...

Figure 5. PuriHcation of y-BBlI by phenyi sepharose HP chromatography. Anaiysis of column fractions by silver stained SDS-PAGE with the corresponding y-BBH activity profile.

Bgure 5. Silver-stained SDS-PAGE of cr tal of die P subunit (Lane 2).purified P subunit preparation (Lane 1)... 

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. 

Figure 1. a - SDS-PAGE of fraction T (silver staining), lane 1 fraction T, lane 2 ovalbumine and lane 3 molecular weight standards. 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Neukirchen et al. (1982) from the Max-Planck Institute employed a similar miniaturized IEF/SDS-PAGE system that was roughly 2 cm x 2 cm. Silver staining was used to detect spots containing as little as 10 pg of protein, and electrophoresis was used to separate the proteins contained within a single Drosophila egg. 

Fractions of 1 CV or less may be collected across the gradient. Fractions from the main product peak and from each side can be analyzed by SDS-PAGE with silver stain. Contaminants can then be identified by their molecular weights. Extrapolating the eluting salt concentration at peak center for each component of interest will allow you to map their relative retention characteristics as a function of both pH and salt concentration. 

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. 

Examine by SDS-PAGE (2.2.31). The test is performed under both reducing and non-reducing conditions, using resolving gels of 14% acrylamide and silver staining as the detection method. 

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