Denaturing polyacrylamide

Middle panel Cell wall proteins were isolated, 10 pgm of each resolved by non-denaturing polyacrylamide gel electrophoresis and PGl and PG2 isoforms detected by activity staining. 

Fig. 8 Autoradiograms of a denaturing polyacrylamide gel for photooxidation of duplex 35/36 in the presence of BamH I. ODNs 35 (a) and 36 (b) were separately 5 -32P-end labeled and hybridized to a non-labeled complementary strand. Lane 1, Maxam-Gilbert G+A sequencing reactions lane 2, in the absence of BamH I lanes 3-5, BamH I. ODNs in lanes 2-4 were irradiated at 312 nm. All samples except in lane 2 were heated with piperidine. The BamH I site and dCNBPU (X) are shown in bold face. For clarity, the autoradiogram for ODN 36 is shown upside-down...

Resuspend sample and detect protected probe fragments by denaturing polyacrylamide gel electrophoresis and autoradiography. 

Figure 3 Biosynthesis and purification of 90-kD elastin analogue analyzed by denaturing polyacrylamide gel electrophoresis (10-15% gradient, visualized by silver staining). Lanes 1-7 time course of target protein expression at 0, 30, 60, 90, 120, 150, and 180 minutes after induction. Lane 9 soluble lysate of induced E. coli expression strain BLR(DE3)pRAMl. Lanes 10-13 protein fractions obtained from immobilized metal affinity chromatography of the lysate on nickel-NTA agarose (imidazole gradient elution). Lanes 8,14 protein molecular weight standards of 50, 75, 100, and 150 kD.

The RNA purification step is necessary for removal of DNA, unincorporated nucleotides, and transcription byproducts. DNA must be removed because it can obstruct subsequent reverse transcription and PCR (Section 7.3.1.6). This can be achieved by DNase I digestion and/or by denaturing polyacrylamide gel electrophoresis (PAGE, 6%-10 % polyacrylamide, 7 M urea) [14]. Unincorporated nucleotides and transcription byproducts are best removed by denaturing PAGE. 

In the Forward reaction, which is analogous to the Maat and Smith procedure, the incubation is carried out in the presence of all four dNTPs and an adjusted concentration of one ddNTP. Under these conditions the exposed 3 -ends, formed by nicking, are elongated in the 5 - 3 direction until polymerization is halted by the incorporation of the dd-nucleotide present in the reaction mixture. The samples generated by the four Backward and four Forward reactions are finally denatured and electrophoresed on a denaturing polyacrylamide gel. The principle of the method is shown in Fig. 3.13. (modified from Seif et al. 1980) and a sequencing gel in Figure 3.14. 

Deproteinized RNA extracts of the organism under study are incubated in vitro with [a-32P]GTP, then analyzed for occurrence of radioactively labeled RNAs by denaturing polyacrylamide gel electrophoresis (adapted from Garriga and Lambowitz8). 

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 22.3. Separation and identification of different cleavage products at the AP site. (A) Duplex oligonucleotide containing an AP site. The damaged strand is labeled by 32P at its 5 end, as indicated by an asterisk. The AP site is designated in the DNA sequence by the X. (B) Schematic illustration of a gel showing the different mobihty of the various cleavage products at the AP site. The various products contain a different 3 end on the labeled 5 DNA fragment. These products are separated by electrophoresis on a 20% denaturing polyacrylamide gel, and the products are visualized by autoradiography.

Figure 19. Bacillus thuringiensis crystal toxin protein. Isolated protein crystals were solubilized as indicated and analyzed by denaturing polyacrylamide gel electrophoresis. Solubilization in Ellis buffer yields the 134,000 dalton protoxin.

Its relative electrophoretic mobility in denaturing and non-denaturing polyacrylamide gels indicated that it had a dimeric structure with an estimated molecular mass of 65kDa per monomer (Figure 11.1). This was confirmed by... 

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