SDS-PAGE gel

Figure 4-5. Two-dimensional lEF-SDS-PAGE.The gel was stained with Coomassie blue. A crude bacterial extract was first subjected to isoelectric focusing (lEF) in a pH 3-10 gradient. The lEF gel was then placed horizontally on the top of an SDS gel, and the proteins then further resolved by SDS-PAGE. Notice the greatly improved resolution of distinct polypeptides relative to ordinary SDS-PAGE gel (Figure 4-4).

Figure 2.1. Schematic illustration oftwo-dimensional gel electrophoresis. Proteins are extracted from the organism of interest and solubilized. The first dimension separates proteins based on isoelectric point. The pi strip is reduced and alkylated and applied to an SDS-PAGE gel for separation by molecular weight. Proteins canbe visualized using a number of staining techniques.

Sweedler reported a two-dimensional separation, where fluorescein thiocarbamyl derivatives of peptides were separated by capillary zone electrophoresis in the first dimension (Liu and Sweedler, 1996). The outlet of the capillary was wiped across the top of an SDS-PAGE gel, where peptides were then separated based on their size. 

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.

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... 

A method using the combination of zinc and imidazole produces similar, negatively stained SDS-PAGE gels.56 Zinc imidazolate forms precipitates in gels except at the sites where precipitation is inhibited by SDS-protein bands.57 The resultant gels are opaque white with clear regions at the sites of the protein bands. They too are best viewed with the gel on a black surface. 

Pore-gradient SDS-PAGE gels can also be used to estimate molecular masses. In this case, log Mr is proportional to log (%T). With linear gradients,%T is proportional to distance migrated, so that the data can be plotted as log Mr vs. log (migration distance). 

Kundu et al.64 used MEKC conditions to assess the purity of two recombinant proteins a cytomegalovirus-CMP-KDO synthetase fusion protein expressed in E. coli and a hepatitis C viral protein expressed in CHO cells. Proteins were prepared in a 10-mM Tris-1% SDS buffer (pH 8.5) and analyzed in a 10-mM borate-100-mM SDS buffer (pH 9.5) in uncoated capillaries. The level of impurities, which varied with the method of protein production, agreed within 5% with results obtained by densitometric scanning of SDS-PAGE gels of the same materials. 

As with SDS-PAGE gel methods, gel-based isoelectric focusing (lEF) methods have been used for decades to determine isoelectric point pi), which is an intrinsic property of protein molecules. Some complex proteins have multiple charge isoforms with multiple isoelectric points. These isoforms are separated as multiple bands in the lEF gel method. However, like other gel method, the lEF gel has limitations it is not automated, not reproducible, and not quantitative for pi determination. It is also labor intensive and requires large volumes of toxic reagents for staining. 

Run an SDS-PAGE gel on the fractions and pool those containing the protein of interest. 

Reference solution (f). Use a solution of molecular mass standards suitable for calibrating SDS-PAGE gels in the range 15-67 kDa. 

The SDS-PAGE gel is agitated for few minutes in Soln. B and then put onto the membrane. Some layers of filter paper soaked in Soln. C finish the sandwich. The blotting apparatus is covered with the cathode plate and the electrodes are plugged into the power supply. Electrotransfer is performed with a constant current of 0.8 mAlcrc of gel area for 0.5-2 h at RT. The voltage does not exceed 15 V. 

The second dimension of 2-DE is SDS-PAGE. Laemmli buffer systems and IPG gel strips are usually preferred. The strips are applied onto the surface of horizontal or vertical SDS-PAGE gels. 

Fig. (1). The wound response in tomato plants. Tomato plants respond to wounding with the transcriptional activation and accumulation of Systemic Wound Response Proteins including defense proteins, proteolysis-associated proteins, signaling-associated proteins, and proteins of yet unknown function in plants defense. The change in gene expression can be monitored on SDS-PAGE gels. In comparison to control plants (I), treatment with systemin (II), or overexpression of the prosystemin cDNA (III) leads to the accumulation of SWRPs (arrowheads) and the downregulation of other, unidentified proteins (triangles). The figure was modified after [13].

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