Nondenaturing gels

An EMSAs is a simple assay to determine if a DNA sequence is able to bind a specific protein. Purified, cellular, or nuclear protein extract is incubated with labeled double-stranded oligonucleotide DNA probes, usually 20-25 nucleotides long. Each probe contains the SNP nucleotide (one per probe) and the identical sequence corresponding to the sequence around the SNP. The mixture is then run on a nondenaturing gel. If the DNA sequence binds the protein, there is a shift in the mobility of the complex (e.g., lane 2, Fig. 12.3). Specific and nonspecific competitors are often added to demonstrate specificity of the shift (lanes 5-7, Fig. 12.3). If an antibody... 

Rafii, F. Cerniglia, C. E. (1990). An anaerobic nondenaturing gel assay forthe detection of azoreductase from anaerobic bacteria. Journal of Microbiological Methods, 12, 139-48. 

Mohamadi et al. [96] reported online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody on-chip coupled to isotachophoresis. The sample injection, preconcentration, and separation were carried out continuously and controlled by a sequential voltage switching program. Preconcentration was carried out with on-chip nondenaturing gel electrophoresis in methylcellulose solution. Furthermore, the authors applied this method for immunoassay of HSA. The separation of HSA and its immunocomplex was achieved in 25 seconds in 1 cm of the microchannel with induced fluorescence detection at 7.5 pM. 

In the second step, 5 fiM of the annealed long strand duplexes were hybridized with 2 fiM short strand at 38 °C for 30 min. Complete hybridization (>98%) was confirmed by nondenaturing gel electrophoresis. 

A proportion of the DNA strands being sequenced have been nicked at a unique position, probably due to some variable specificity or contaminant in the restriction enzyme. Such breaks are not revealed when the double-stranded fragment is electro-phoresed on a nondenaturing gel... 

Renatured RNAs can be examined for gross conformational homogeneity by nondenaturing gel electrophoresis in either polyacrylamide gels or Nusieve agarose gels. Electrophoresis is carried out in TBE supplemented with 5 mM MgCl2 and 50 mM KC1 at 4 V/cm at room temperature. 

Fig. 3. Only CCT (but not GroEL) can productively fold actin or tubulin. Binary complexes formed in vitro between GroEL and unfolded /bactin (A, B) or a-tubulin (C) were incubated in the presence of GroES, ATP (A, B) or ATP, GTP and tubulin-specific chaperones (C), and an eightfold molar excess of CCT (B, C). After the incubation times (in minutes) shown, the reaction products were analyzed by nondenaturing gel electrophoresis. Arrows (top to bottom) show the location of CCT binary complexes, GroEL binary complexes, and either native actin or native tubulin heterodimers. Adapted from Tian et al. (1995b), with permission.

Figure 3 (A) Nondenaturing gel analysis of Pu27. (B) DMS footprinting (lanes 4-6) of...

Many published protocols for mRNA differential display entail the use of denaturing polyacrylamide gels for the separation of the PCR-amplified bands (5,6,7) The use of nondenaturing gels avoids the problem of multiple banding patterns caused by the tendency of Taq and other DNA polymerases to add a single dTTP residue to the 3 end of amplified DNA sequences (14,15). 

Characterization of the purified protein by nondenaturing gel filtration reveals a native molecular mass of approximately 400 kDa. SDS-PAGE analysis of the pure enzyme shows a single band with a molecular mass of about 52 kDa. This indicates that the l-aminopeptidase most probably is a homo-octameric enzyme. The isoelectric point of the protein is estimated as pH 10.5 using an isoelectric focusing (lEF) 3-9 Ph tgel with an expanded pH range. 

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