Native gel electrophoresis

In native gel electrophoresis and CZE, the sample components are resolved by their differences in electrophoretic mobility or mass-to-charge ratios. Electrophoretic analysis under native conditions in gel electrophoresis is not as widely used as SDS-PAGE. In gels, disadvantages of native analysis are the low field... 

Ledoux, D.N., Be, X.H. and Singh, B.R., Quaternary structure of botulinum and tetanus neurotoxins as probed by chemical cross-linking and native gel electrophoresis, Toxicon, 32, 1095-1104, 1994. 

Figure 9.2 Native gel electrophoresis of the Tetrahymena P4—P6 RNA. (A) The folded and extended forms equilibrate rapidly, producing a single band whose mobility reflects the average structure of the RNA. U1, U2, BP5/5a refer to mutations in a hinge region. RNAs were run on native 6% (19 1 monorbis) polyacrylamide gel in TBE + 10 mM MgCl2 at 25 C. (B) Ferguson plot shows that the relative mobility (M) depends linearly on gel concentration, as predicted by the Ogston model. Reprinted from Szewczak and Cech (1997).

We probed the reactivity of the carboxylates using a fluorescent carboxylate-selective chemical dye, N-cyclohexyl-N -(4-(dimethylamino)naphthyl)carbodiimide (NCD4). Using UV-visible spectroscopy, native gel electrophoresis, and denaturing gel electrophoresis, covalent modification with the dye was confirmed (Figure 9.5). The latter showed that the dye was attached to both the S and the L subunit, an expected observation, as the structural data suggested carboxylates on both subunits. A quantification of the number of bound dye molecules could not be achieved owing to the instability of the dye in aqueous solvent a reliable extinction coefficient could not be determined. 

After chemical derivatization, the integrity of the particles was confirmed by transmission electron microscopy (TEM), dynamic light scattering, and native gel electrophoresis. Measurements of the particle size by dynamic light scattering showed that the ferrocene-decorated CPMV particles (CPMV-Fc ) had an increase in radius of about 0.7 nm which is in good agreement with the size of the ferrocene moiety. 

In detail, viral wild-type particles of one set were labeled with the fluorescent dye AlexaFluor (AF) 488 and biotin both groups were attached to surface available lysines. Another batch was labeled with AF568 and biotin, also at addressable lysines. Both types of building block, in the following referred to as CPMV-biotin-AF488 and CPMV-biotin-AF568, were characterized by TEM, UV-visible spectroscopy, native gel electrophoresis, and dot blot studies. TEM studies verified that the particles remained intact after chemical modification. UV-visible spectroscopy confirmed covalent modification and also allowed quantification of the number of labels per particle the particles displayed around 40 biotin moieties and around 200 dyes. 

The purity of the CL mutant protein expressed by yeast is further verified by denaturing polyacrylamide gel electrophoresis with SDS26 and native gel electrophoresis.27 SDS stacking (5% top gel and 15% bottom gel) polyacrylamide gel and Laemmli gel buffers are used. Ten percent native... 

FIGURE 8. Determination of the native molecular weight of the purified PSPBP from Acanthocardia tuberculatum foot by native gel electrophoresis of various concentrations of polyacrylamide (6, 8, 10 and 12 %). Molecular weight marker proteins were commercial tetrameric urease (545 kDa), dimeric urease (272 kDa), dimeric BSA (132 kDa), monomeric BSA (66 kDa) and ovalbumin (45 kDa) (Sigma). (A) represents the relative mobilities of proteins plotted as Log (Rf x 100) vs. gel concentration. A plot of the obtained slopes vs. molecular weight was linear and used to determine native PSPBP molecular weight (B). 

The anthozoan protein amajCFP as well as its mutant amajCFP-II (see table 5) form tetramers as judged by semi-native gel-electrophoresis [55]. For the anthozoan CFPs protein oligomerization and aggregation needs to be investigated. 

Reflect and Apply When proteins are separated using native gel electrophoresis, size, shape, and charge control their rate of migration on the gel. Why does DNA separate based on size, and why do we not worry much about shape or charge ... 

LDH 3 has the subunit composition H M. Each of the subunits is coded for by a separate gene, so in order to clone LDH 3, one would have to clone the gene for the M subunit and the gene for the H subunit. These would be separate cloning experiments. Each gene would be cloned into an expression cell line, and the proteins would be expressed. The individual subunits could then be combined, and they would form tetramers, some of which would be LDH 3. This could be verified by native gel electrophoresis. 

A number of separation modes are possible. In native gel electrophoresis, charged analytes are distinguished according to their apparent mobility, Papp, and size. In sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analytes are treated such that they all exhibit the same charge to size ratio. Hence, they are separated only by differences in their size. This method can be used for... 

Separation gel without SDS for the SDS electrophoresis No problem The SDS from test buffer and running buffer rrms faster than the proteins (i.e., they remain in an environment that contains SDS). The advantage of the SDS-free separation gel with SDS-free test buffer and running buffer, the Ahn system becomes a native gel electrophoresis. 

Some proteins (e.g., membrane proteins) aggregate imder the conditions of lEF. Then you get the same smeary result as native gel electrophoresis (see Section 1.3.2). Here it sometimes helps to dissolve the protein sample in SDS. However, because SDS protein complexes do not focus they do not have a useful isoelectric point. You have to push out the SDS again after diluting with NP-40 or CHAPS (Garrels 1979 Corbett et al. 1994). Many membrane proteins cannot be impressed with this trick and form aggregates again after NP-40/urea is added. They remain only in solution as SDS protein complexes or as complexes with other charged... 

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