Non-denaturing gel electrophoresis

Non-denaturing gel electrophoresis Denaturing (SDS) gel electrophoresis Two-dimensional electrophoresis Capillary electrophoresis Peptide mapping HPLC (mainly RP-HPLC)... 

The extent of ADP-ribosylation is checked by separating modified and unmodified actin using non-denaturing gel electrophoresis, whereas SDS gel electrophoresis is not efficient. ADP-ribosylation changes the isoelectric point of actin to more acid values, resulting in increased migration. This method is also appropriate to study double or triple ADP-ribosylation of actin, e.g., in the presence of iota toxin and/or turkey erythrocyte transferase (Just ef al., 1995). The latter transferase modifies actin even at two arginine residues. 

Several controls may be performed to confirm that the folded structure indeed corresponds to a quadruplex. The cation dependency of its stability (which follows a K > Na > Li preference order) is a good indieation. One may also demonstrate that the thermal difference spectra or circular dichroism spectra of all these structures are in agreement with the formation of quadruplexes. Furthermore, non-denaturing gel electrophoresis may confirm that a single, major retarded (for hi- and tetramolecular) band is observed when the oligonue-leotides are pre-incubated at high concentration in the presence of NaCl or KCl. 

Becker, J. Su., Mounicou, S., Zoiiy, M. V., Becker, J. S., Lobinski, R (2008) Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Talanta, 76, 1183-1188. 

Figure 55. Charactetization of the p-Ribo(A8)/jp-Ribo(U8) duplex by temperature dependent UV-spectroscopy, temperature dependent CI>-spectro-sct y and (non-denaturing) gel electrophoresis [8].

Figure 2 (left). Permeation chromatography of ESS on Bio-Gel P-2 column. Three major peaks were observed. All of the chemoattractive activity for snakes was in Peak 1, none in peaks 2 or 3. Pe 3 contained alarm pheromone for earthworms. Figure 3 (right). Polyacrylamide gel electrophoresis of ESS (B and D), purified chemoattractant (A) on non-denaturing gel protein markers (E) and purified chemoattractant (F) on SDS-denaturing gel. From Jiang et al. (1990) with permission. 

Rothe, GM, Determination of Molecular Mass, Stoke radius. Frictional Coefficient and Isomer-Type of Non-denatured Proteins by Time-Dependent Pore Gradient Gel Electrophoresis, Electrophoresis 9, 307, 1988. 

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. 

After maximum radioactivity incorporation the protein is denatured and generally subjected to HPLC or gel-electrophoresis. Those methods separate the proteins from the specific tissue by size and the radioactivity distribution can be determined among the protein components. The specifically labeled biopolymers are distinguished simply by a competition experiment performed by the addition of excess of non-labeled parent ligand. It eliminates the radioactivity incorporation. 

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