SDS-electrophoresis

Two-dimensional gel electrophoresis (2DE) is a two-dimensional technique for protein separation, which combines isoelectric focusing and sodium dodecyl sulphate (SDS) electrophoresis. The high resolving power results from separation according to charge (isoelectric point) in the first dimension and size (mobility in a porous gel) in the second dimension. Depending on the gel size, from several hundred to more than 5,000 proteins can be separated. 

The 5-iodoacetamido derivative of fluorescein (5-IAF) has been used to label numerous proteins and other biomolecules, including actin (Plank and Ware, 1987), myosin (Aguirre et al., 1986), troponin (Greene, 1986), hemoglobin (Hirsch et al., 1986), and sulfhydryl-containing proteins separated by SDS electrophoresis (Gorman, 1984). 

The reagent is similar to another maleimide-containing biotinylation reagent, 3-(N-maleimi-dopropionyl) biocytin, a compound used to detect sulfhydryl-containing molecules on nitrocellulose blots after SDS-electrophoresis separation (Bayer et al., 1987). Biotin-BMCC should be useful in similar detection procedures. 

Analyze the quenched reaction by SDS electrophoresis, Western blotting, and mass spec analysis. 

Quench the cleavage reaction by the addition of an equal volume of SDS electrophoresis sample buffer containing up to 40 percent glycerol. The SDS will denature the protein interaction and glycerol acts as a free radical scavenger, thus effectively quenching the reaction. 

Immediately after irradiation, stop the reaction by the addition of 7 pi of 4 X SDS electrophoresis loading buffer or the equivalent (with a high concentration of reducing agent present) 0.2M Tris, 8 percent SDS, 2.88M P-mercaptoethanol, 40 percent glycerol, 0.4 percent xylene cyanol, 0.4 percent bromophenol blue. Heat the sample at 95°C for 5 minutes and analyze the complexes formed by electrophoresis. 

Figure 5.2 The production and purification of tPAfrom the milk of transgenic goats. (WAP promoter murine whey acid promoter). The downstream processing procedure yielded in excess of an 8000-fold purification factor with an overall product yield of 25 per cent. The product was greater than 98 per cent pure, as judged by sodium dodecyl sulfate (SDS) electrophoresis...

The various types of capillary electrophoresis are performed either in free solution or in gels. The choice of method depends on the nature of the sample and the analytical objective but capillary gel electrophoresis, including iso-electric focusing and SDS electrophoresis, is particularly useful for protein applications. 

Figure 11.14 Determination of the relative molecular mass (RMM) of a protein by SDS electrophoresis.

Check the proper connection by wires In SDS electrophoresis the migration is (mostly black wire) -I- (mostly red wire), when cetyltrimethylammonium bromide (CTAB) is used instead of SDS the direction is -I- ... 

Combining isoelectric focusing and SDS electrophoresis sequentially in a process called two-dimensional... 

To determine molecular weights of proteins by gel electrophoresis, a protein must be rendered into the random coil conformation by breaking disulfide bonds (mercaptoethanol) and physical bonds (urea), and the charge must be made overwhelmingly negative with SDS. Electrophoresis may be performed at a neutral pH. 

Usually, immunoglobulins are N-glycosylated at the Fc part in some cases, terminal neuraminic acid may be missing. This leads to slightly different isoelectric points of the various members in the family called isoforms. Therefore, an IgG preparation which exhibits a single band in sodium dodecyl sulfate (SDS) electrophoresis exhibits a characteristic band pattern in isoelectric focusing (Fig. 4). 

Figure 3. SDS-Electrophoresis of ADPGlc-synthetase photoinactivated with [1 C] 8-N3ADPGIC. 1.8 yM (subunits) enzyme in 20 mM HEPES, 1 mM 3 PGA, 5 mM MgCl2 and 1.5 mM C1 C] 8-N3ADPGIC (specific activity, 1.8 x 107 cpm per ymole), was irradiated 10 min with UVS-51 lamp from a distance of 5 cm. Lane 1 - unirradiated control Lane 2 - enzyme...

The prenyl transferase from avian liver has been crystallized,40 and was found to be a dimer of molecular weight 86 000 dalton the subunits could not be resolved by SDS electrophoresis. The enzyme catalysed the formation of FPP from IPP and either DMAPP or GPP, and this was accompanied by the synthesis of small amounts of geranylgeranyl pyrophosphate (GGPP). This is the first stable crystalline enzyme of the steroid and terpenoid pathways to be prepared. 

As above, but all proteins are made -ve by SDS and so separate according to their size Proteins migrate through gels to their isoelectric point (pH where they have no net charge) Combines isoelectric focusing and SDS-electrophoresis samples are run in two directions to increase resolution... 

In the first dimension the proteins are focused according to their isoelectric point [isoelectric focusing (IEF) and subsequently in the second dimension according to their molecular size (SDS electrophoresis or SDS-PAGE). 

Apart from the wide ran of staining techniques, fluore nt and radioactive labels are often used. FITC is used for protein detection following SDS-electrophoresis, e.g. Rat hemoglobin fractions have been assayed by labeling with Double labeling with and makes it possible to detect proteins from two different... 

The use of SDS is not always without drawbacks. One of the most important is encountered when the sample is rich in DNA. A terrible viscosity results, which can hamper the electrophoresis process. Moreover, some protein classes (e.g. glycoproteins) bind SDS poorly and are thus poorly separated in the subsequent electrophoresis. In such cases, it is advisable to use cationic detergents. They are usually less potent than SDS, so that a urea-detergent mixture must be used for optimal solubilization (MacFarlane 1989). Moreover, electrophoresis in the presence of cationic detergents must be carried out at a very acidic pH, which is not technically simple but still feasible (MacFarlane 1989). This technique has however gained recent popularity as a double zone electrophoresis method able to separate even membrane proteins (Hartinger et al. 1996), and showing more separation power than SDS electrophoresis alone. 

A few practical notes should be emphasized regarding SDS electrophoresis. Potassium salts should be avoided and sodium salts used in their places because potassium dodecyl sulfate is quite insoluble. In addition, even sodium dodecyl sulfate is insoluble below about 10°C. Although not specifically cited above, disc gel electrophoresis may also be used in the presence of SDS. These techniques, described by Studier (11) and Ames (12), are of great advantage when the sample volume is in excess of 10 to 20 /Ltliters per gel. A widely used buffer system for SDS acrylamide gel electrophoresis is that developed by Laemmli (41). 

S. acidocaldarius (DSM 639) extrudes protons and synthesizes ATP while respiring endogenous substrates [65]. DCCD inhibits this ATP synthesis and the inhibitor binds to a membrane proteolipid [57]. This proteolipid (M, 34 000) can be purified using procedures for isolating DCCD-binding proteolipids [66]. Two subunits are observed after SDS-electrophoresis (M, 17 000 and 19000) but a single peptide (M, 6000) is detected when SDS-electrophoresis is carried out in the presence of 8 M urea. The amino-acid sequence from the N-terminal end of an Mr 2000 CNBr-peptide exhibits considerable homology to sequences of subunit c from the Fq of E. coli and the thermophilic bacterium, PS3 [64]. 

Amino acid analysis is often touted as the most accurate method for determination of protein concentration. The data from this 1996 ABRF AAA study indicate that the vast majority of member facilities that participated in this study quantitate soluble protein well. The most striking aspect of this study, however, was the ability of the laboratories to identify the protein solely on its amino acid composition. The data from approximately 90% of the participants were sufficient for correct identification, if one knew the species of the protein s origin. Currently, identification of unknown proteins from AAA data is not frequently used for simple soluble proteins, such as triosephosphate isomerase. The technique is more commonly used to identify proteins that have been separated by two dimensional analysis on isoelectric focusing and SDS electrophoresis and then transferred to PVDF membranes. Such samples are usually present in low... 

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