Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE

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. 

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. 

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of <100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

The boiled samples are resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 4 to 20% polyacrylamide gels, loaded in the following order 3% input —10 fA of eluate (15% of the total volume of the boiled eluate El) —20 fA of eluate (30% E2) —3% supernatant, respectively, and transferred to nitrocellulose membranes (Novex or Bio-Rad). 

Various methods have been used to examine the composition of proteins adsorbed to SAMs. Overall adsorption patterns can be examined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [50, 76, 77]. Absorbed proteins are eluted from the surface with surfactant (SDS), and then separated by electrophoresis. The proteins of interest are examined by western blotting [50, 76, 77]. Protein-specific antibodies can be used to detect proteins of... 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

Figure 6.5. Immunoblot of the urease large subunit. Extracts of H. pylori wild type (WT) and a hypA mutant from cells grown without nickel supplementation were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by blotting with an anti-urease large-subunit antiserum. Urease activity was 58pmolmin mg for the wild type and Opmolmin" mg for the hypA mutant.

According to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and CaM affinity chromatography analyses, compounds 44—53 interacted with both spinach and bovine-brain CaMs. In order to quantify the interaction of the phytotoxic agents 44—53 with... 

PCR and in vitro recombination reactions are quite simple and straightforward for generating multiple expression plasmids in parallel, e.g., in a 96-well plate see Fig. 3a). The first preliminary expression experiment was done to evaluate the production level of each GST-fused protein. In this step, we compared the staining patterns of E. coli proteins harboring expression plasmids with the patterns of proteins harboring empty vectors on sodium dodecyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the same culture conditions see Fig. 3b). In addition to... 

A fully automated two-dimensional electrophoresis (2DE) system for rapid and reproducible protein analysis is described. 2DE that is a combination of isoelectric focusing (lEE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein expression analysis. Here, all the operations are achieved in a shorter time and all the transferring procedures are performed automatically. The system completed the entire process within 1.5 h. A device configuration, operational procedure, and data analysis are described using this system. 

Two-dimensional electrophoresis (2DE) that is a combination of isoelectric focusing (lEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (4) is widely used for protein expression analysis. 

One of the most widely used forms of gel electrophoresis is known as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide gel has several advantages that account for its extensive use. It has minimal adsorptive properties and produces a negligible electroosmotic effect (Hjelmeland and Chrambach 1981). In identity tests, for the determination of molecular weight, SDS-PAGE has been shown to be an appropriate, fast, and easy method that is often used in quality control laboratories. The use of SDS-PAGE followed by a densitometric analysis, such as MS, is a helpful technique for the determination of peptide or... 

It is unlikely that the protein fractions from this experiment contain a single type of protein. How many different proteins are present What is the relative abundance of each protein Is a-lactalbumin the predominant protein in the isolated fractions What are the approximate molecular weights of a-lactalbumin and other proteins These questions may be answered by analysis of the isolated fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see Chapter 4). The technique of SDS-PAGE will be introduced and applied to the column-purified fractions, crude whey fraction, and standard a-lactalbumin. 

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... 

The great analytical power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) makes it one of the most effective tools of protein chemistry and molecular biology. In the past, there have been many attempts to convert the technique from analytical to preparative scale, because by SDS-PAGE, one can resolve more than 100 protein species in 5—6 h. The number of papers that describe preparative elution from polyacrylamide gels is immense (for example, see refs. 1—5). In spite of the numerous variations m the procedure of elution, none of the available methods is entirely satisfactory. Some of the methods are very laborious, and others lead to loss of resolution or poor recovery. 

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