Two-dimensional SDS-PAGE

The best method to determine the success of antibody production to the minor components was made by two dimensional SDS-PAGE and immunoblotting (24). A comparison of the antisera (day 112 antisera) from the three groups demonstrated that the cascade immunization antisera detected a number of minor components (Figure 4C, arrows) which were not observed with the conventional or passive antisera (Figure 4B and D). It was clear from these results that the cascade antisera was far superior in its spectrum of antibody reactivity and, in fact, was comparable or superior in detection of ECPs to silver stain (Figure 4A). Although silver stain appeared to have an improved detection of certain low MW or basic proteins,... 

Principle of the two-dimensional SDS-PAGE and immobilised pH gradient gels... 

Box 12.1 Tagging and Resolving by One- and Two-dimensional SDS-PAGE of Proteasomes by Activity-based Protein Profiling... 

The procedure is schematically illustrated in Fig. 3. Isolation of proteins separated by one or two-dimensional SDS-PAGE followed by electrotransfer onto a support compatible with direct sequence analysis has become the method of choice for the isolation of proteins from complex protein mixtures for N-terminal sequencing. The following describes an optimized protocol. 

Protein samples are separated by one-dimensional SDS-PAGE or two-dimensional gel electrophoresis in polyacrylamide gels. The separated proteins are then transferred (blotted) to a nitrocellulose or nylon sheet. This is incubated with specific antibody to the protein and then unbound antibody is washed away. Those proteins in the gel that bind the antibody are detected either by autoradiography (if the specific antibody was radiolabeled) or by using a second labeled antibody that binds to the primary antibody. 

Gupta, R.B., Shepherd, K.W. (1990). Two step one dimensional SDS-PAGE analysis of LGM subunits of gjutenin. 1- Variation and genetic control of the subunits in hexaploid wheats. Theor. Appl. Genet., 80, 65-74. 

Two-dimensional BAC/SDS polyacrylamide gel electrophoresis has been established as further tool in the field of proteome research, especially regarding the separation and analysis of membrane proteins. It is by far more efficient in resolving membrane proteins than common 2-DE and furthermore can be utilized in a complementary way to one-dimensional SDS-PAGE. Therefore, among other techniques, future proteome studies focussing on membrane proteins should include 2-DB as well. 

Figure 10.3 One-dimensional SDS-PAGE separations of glutenin subunits from six wheat cultivars. The one-step procedure (a) with and (b) without alkylation (c) the second step of the two-step method. The positions of MW markers are shown on the left side of the figure. (Gupta, R. B., and F. MacRitchie, F. 1991. Journal of Cereal Science 14 105-109.)...

Hgure 9 Canine urine. One-dimensional SDS-PAGE electrophoresis of the proteins in dog urine from two different animals before and after castration. After castration the pattern is similar to that of females. Differences in levels of the male-specific protein (indicated by an arrow) may be associated with different prostate disorders. (Courtesy of Miller I, University of Veterinary Medicine, Vienna.)... 

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).

Poehling reported a microscale two-dimensional gel electrophoresis system 25 years ago (Poehling and Neuhogg, 1980 Neuhoff, 2000). In that system, separation in the IEF dimension was performed in thin, gel-filled tubes. After IEF, the gel was transferred to a 3 cm x 3.5 cm polyacrylamide gel, where proteins were separated by SDS-PAGE. Several hundred components were resolved from a few micrograms of protein homogenate. 

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. 

One concern relating to SDS-PAGE-based purity analysis is that contaminants of the same molecular mass as the product will go undetected as they will co-migrate with it. Two-dimensional electrophoretic analysis would overcome this eventuality in most instances. 

Phosphorus, fatty acids, carbohydrates, glycerol, and amino acids were analyzed by the method described in our previous paper [8] and references cited therein. SDS-PAGE [8], TLC [9], HPLC [9], determination of phos-phomonoester [8], reducing sugar analysis [13], methylation analysis [14], and hexose analysis [15] were performed as described in the respective literature. Two dimensional TLC was performed on silica-gel plate (Merck Silicagel 60 F254 No. 5715) using the solvent systems, chloroform-methanol-acetic acid (65/10/1, v/v/v) for the first development and chloroform-methanol-25% ammonia solution (65/10/1) for the second. 

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