Proteins SDS-PAGE

X 33000, recombinant protein, SDS-PAGE, x 328000, deduced from gene sequence, 14 residue C-terminal sequence is important for enzyme activity [14]) [14]... 

In Experiment 4, your sample of a-lactalbumin extracted from bovine milk was subjected along with other proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After staining with the dye Coomassie Blue, deeply colored bands appeared on the gel wherever there was a protein. You suspected that some of the blue bands on the gel were due to a-lactalbumin. If molecular weight standards were included on the slab gel, you were able to estimate the molecular weight for a-lactalbumin and other proteins. SDS-PAGE is indeed a very effective analytical tool to achieve fractionation of protein mixtures, to analyze purity, and to estimate molecular weight, but it provides no experimental data to prove the identity... 

Application 2 Capillary Gel Electrophoresis. Recently, Karger and co-workers demonstrated the use of polyacrylamide gel-filled capillaries to separate peptide/protein (SDS PAGE) Qfi) and oligonucleotide mixtures (12,22) by capillary electrophoresis. This mode of CE operation may prove to couple well with on-line radioisotope detection. The results of several preliminary capillary electrophoresis separations using gel-filled capillaries and on-line radioisotope detection using the coincidence unit described here are presented below. 

SDS-CGE Molecular size Estimation of relative molecular mass of protein (10-200kDa) and purity and stability of peptides and proteins SDS-PAGE... 

Figure 2 shows that most of the dehydrase activity is eluted when Triton X-100 is present in the running buffer. The protocol of purification results in an average of 16-fold purification with a recovery of 50 % of the total activity. The final concentrated fraction has a specific activity of 45 /imoles/min.mg protein. SDS-PAGE characterization shows that the fraction contains two major proteins of 67 and 55 kDa (data not shown). 

The reaction center is built up from four polypeptide chains, three of which are called L, M, and H because they were thought to have light, medium, and heavy molecular masses as deduced from their electrophoretic mobility on SDS-PAGE. Subsequent amino acid sequence determinations showed, however, that the H chain is in fact the smallest with 258 amino acids, followed by the L chain with 273 amino acids. The M chain is the largest polypeptide with 323 amino acids. This discrepancy between apparent relative masses and real molecular weights illustrates the uncertainty in deducing molecular masses of membrane-bound proteins from their mobility in electrophoretic gels. 

FIGURE 5A.4 A plot of the relative electrophoretic mobility of proteins in SDS-PAGE versns the log of the molecnlar weights of the individnal polypeptides. 

Microtubule-associated proteins bind to microtubules in vivo and subserve a number of functions including the promotion of microtubule assembly and bundling, chemomechanical force generation, and the attachment of microtubules to transport vesicles and organelles (Olmsted, 1986). Tubulin purified from brain tissue by repeated polymerization-depolymerization contains up to 20% MAPs. The latter can be dissociated from tubulin by ion-exchange chromatography. The MAPs from brain can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 

The protein was purified by a dialysis procedure, denatured and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting indicated that the protein of interest consisted of two components, one of which increased in concentration as the purification proceeded. The authors initially suggested that this could be due to the presence of a number of species produced by modification of the amino acid side-chains, for example, by glyco-sylation, or by modification of the C- or N- terminus. 

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) An electrophoretic technique used for the separation of proteins. 

Figure 4-4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein standards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

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).

The number of different proteins in a membrane varies from less than a dozen in the sarcoplasmic reticulum to over 100 in the plasma membrane. Most membrane proteins can be separated from one another using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique that has revolutionized their study. In the absence of SDS, few membrane proteins would remain soluble during electrophoresis. Proteins are the major functional molecules of membranes and consist of enzymes, pumps and channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymatic properties of several different membranes are shown in Table 41-2. 

Analysis by SDS-PAGE Resolves the Proteins of the Membrane of the Red Blood Cell... 

When the membranes of red blood cells are analyzed by SDS-PAGE, about ten major proteins are resolved... 

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