Nondenaturing polyacrylamide gel electrophoresis

Use nondenaturing polyacrylamide gel electrophoresis (PAGE) to separate and visualize LMW-Cd2+-PC2 and HMW-Cd2+-PC2 complexes contained in the crude extract. A detailed protocol for doing this is given by Abrahamson el al.27. The peptides should be visualized by silver staining but referenced to the 109Cd result since proteins other than PCs may be present. 

M. tuberculosis, the katG of M. leprae has been reported to be a pseudogene, implication that isonia-zid, which is activated to a potent tuberculocide agent by catalase, is unlikely to be of therapeutic benefit to leprosy patients. Kang et al. (2001) found catalase-hke activity in M. leprae cell lysates by the diaminobenzidine staining method with nondenaturing polyacrylamide gel electrophoresis. 

Nondenaturing Polyacrylamide Gel Electrophoresis (NPAGE) as a Method for Studying Protein Interactions... 

B. ZYMOGRAPHY OF PROTEASES WITH DEVELOPED FILM AFTER NONDENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS ON PHASTSYSTEM GELS... 

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

Fig. 5.2.10 Nondenaturing two-dimensional electrophoresis with agarose gel electrophoresis in the first dimension and gradient polyacrylamide gel electrophoresis in the second dimension combined with anti-apolipoprotein AI (apoA-I) immunoblotting differentiates apoA-I-contain-ing lipoproteins by charge (pre-/ l-LpA-I versus a-LpA-I) and size (HDL2 versus HDL3). In normal plasma the major part of the HDL pool is formed by a-LpA-I, a smaller by pre-/ l-LpA-I (left picture). In the plasma from Tangier patients only some pre-/ l-LpA-I particles are present (right picture)...

A student isolated an enzyme from anaerobic bacteria and subjected a sample of the protein to SDS polyacrylamide gel electrophoresis. A single band was observed on staining the gel for protein. His adviser was excited about the result, but suggested that the protein be subjected to electrophoresis under nondenaturing (native) conditions. Electrophoresis under nondenaturing conditions revealed two bands after the gel was stained for protein. Assuming the sample had not been mishandled, offer an explanation for the observations. 

The mass of a protein can be directly determined by sedimentation equilibrium, in which a sample is centrifuged at relatively low speed so that sedimentation is counterbalanced by diffusion. The sedimentation-equilibrium technique for determining mass is very accurate and can be applied under nondenaturing conditions in which the native quaternary structure of multimeric proteins is preserved. In contrast, SDS-polyacrylamide gel electrophoresis (Section 4.1.4) provides an estimate of the mass of dissociated polypeptide chains under denaturing conditions. Note that, if we know the mass of the dissociated components of a multimeric protein as determined by SDS-polyacrylamide analysis and the mass of the intact multimeric protein as determined by sedimentation equilibrium analysis, we can determine how many copies of each polypeptide chain is present in the multimeric protein. 

Preparing a Non-Denaturing Polyacrylamide Gel Electrophoresis with an 8% nondenaturing polyacrylamide gel is commonly used and offers fine separation with high resolution of DNA fragments. Table 3.10 includes the components and the quantities to prepare an 8% non-denaturing polyacrylamide gel. 

The bulk proteins of guinea pig seminal vesicle secretion that are soluble in 150 mM NaCl have been separated by ion-exchange chromatography and other techniques including gel or paper electrophoresis under nondenaturing or denaturing conditions (7, 160-166). In sharp contrast to rat vesicular secretion proteins (see below), prior exposure of whole or saline-soluble guinea pig seminal vesicle secretion proteins to thiols does not alter the profiles of bands separable by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate with or without concentrated urea (J. Wilson and H. G. Williams-... 

Perform polyacrylamide gel electrophoresis with 12.5% homogeneous gels under nondenaturing conditions on a PHastSystem. 

Paech, C., Christianson, T., and Maurer, K.-H. (1993b) Zymogram of proteases made with developed film from nondenaturing polyacrylamide gels after electrophoresis. Anal. Biochem. 208, 249-254. 

Analyze the samples by electrophoresis through a 0 4-mm-thick, 6% nondenaturing polyacrylamide gel see Note 10) To the purified PCR sample add 2 pL of DNA loading dye, 4 pL of this sample can then be loaded on a... 

FIGURE 6. Gel electrophoresis of purified E. coli Met(0)-peptide reductase. (A) 15% polyacrylamide containing 0.1% NaDodSO. (B) 13% polyacrylamide (pH8.8) nondenaturing. Reproduced by permission of Academic Press from Brot and coworkers112. 

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