Native gels

Native gel systems do not contain SDS. Thus, the charge of the proteins in the gel is dependent on their isoelectric point and the pH of the buffer used. With native gels, the protein solution is loaded directly on the gel with cane sugar and an indicator, but without pretreatment. In the electric field, some of the proteins then move to the positive pole and some to the negative pole. The speed with which a protein moves depends on its size, its charge, the porosity of the gel matrix, and the pH of separation gel and running buffer. 

Maurer (1971) describes a half-dozen native systems. Native gels have fallen out of fashion, because neither MW nor isoelectric point can be determined and they are not suited for membrane proteins (see, however, the following). They are occasionally used to check the purity of soluble proteins. 

Advantages Many proteins survive the electrophoresis and can be eluded again from the gel in active form. Some enzymes can be detected directly in the gel via the enzyme reaction. 

Problems Running a native gel takes hours. Hydrophobic (integral) membrane proteins smudge in conventional native gels, even if nonionic detergents such as TRITON-X-100 have been added to buffer and gel. 

The blue gel is both a new purification method and potentially a means of determining the quaternary structure of proteins (see Chapter 8), especially in combination with an SDS gel— which, as a second dimension, cleaves the natively separated oligomers into their subimits. 

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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. 

The dye Coomassie Brilliant Blue R250 nonspecifically binds to all the protein. The gel is soaked in the dye for it to seep in and bind to the proteins. The gel is then destained to remove the unbound dye. The dye binds to the protein and not the gel, and hence the protein bands can be visually seen. The binding of the dye to the protein is approximately in stoichiometry, so the relative amounts of protein can be determined by densitometry. For most SDS and native gels, separated proteins can be simultaneously fixed and stained in the same solution. 

To perform gel-shift selection experiments, prepare a native gel, containing 3% acrylamide (stock 38.5% acrylamide and 1.5% bis-acrylamide) and lx TBE, adjusted to pH 7.4. 

In the first dimension, 20 [tl plasma is separated by electrophoresis at 4°C in a 0.75% agarose gel using a 1 2 16 dilution of a barbital buffer. Bromophenol blue is added to a standard sample to visualize albumin in the native gel. The electrophoresis is stopped when the albumin/bromophenol blue marker has migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins are then transferred to a 4-20% polyacrylamide gradient gel. Separation in the second dimension is performed at 40 mA for... 

Native Zone Electrophoresis. In some cases, good resolution between sample species can be obtained with little or no sample pretreatment. In these eases, the gels arc said to he native gels. In this method, the charge on the individual sample component is primarily responsible for its differential migration. 

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).

Alternatively, peaks areas can be defined manually and the entire volume of the peak integrated. This method is more tolerant of imperfect gels. However, because the bands in native gels are often broad, it is critical to set a uniform criterion (such as pixel intensity) for defining peak boundaries and... 

Because samples must be loaded on the gel immediately, the native gel is cast and prerun before the start of the reaction. Folding reactions are set up in a sufficient volume so that aliquots can be withdrawn at various times (20—40 /iL). We obtain the most reproducible results by first mixing all of the reaction components except the RNA. This mixture is warmed to the desired reaction temperature (e.g., 30—50 °C) in a water bath or heating block placed near the native gel apparatus. The folding reaction is then started by adding a 5 X or 10 x stock of unfolded RNA to the folding buffer. 

Buchmueller el al. (2000) measured the mobility of the bI5 ribozyme in native gels containing 0—20 mM MgC. Double-stranded 174 bp RNA,... 

A final concern is the extent to which the proportion of each conformer is faithfully captured by the native gel. Although very small structural differences, such as isomerization within an active site, may not be resolved unless they alter the hydrodynamic profile of the RNA, native PAGE results generally correlate well with other measures of RNA folding in solution. Because 10-30 s are needed for samples to enter the gel, native PAGE is most successful at resolving conformational states that do not exchange within this time (Fig. 9.3A). The entrapment of different conformers is... 

Based on all evidence above, I propose the mode of action of OBPs follows the model depicted in Figure 15.8. OBPs participate in the selective transport of pheromones and other semiochemicals to their olfactory receptors. By selectivity, I do not mean that OBPs are the only filter of the olfactory system. In other words, I believe that OBPs may bind, solubilize, and transport structurally related ligands, but not compounds with unrelated chemical structures. This view is supported by the native gel-binding assay with BmPBP (Wojtasek and Leal, 1999) that showed binding to bombykol, but not to lactones, such as (R)- and... 

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. 

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