Electrophoresis denaturing

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

Fig. 17.6. Southern, Northern, and Western blots. For Southern blots, DNA molecules are separated by electrophoresis, denatured, transferred to nitrocellulose paper (by blotting ), and hybridized with a DNA probe. For Northern blots, RNA is electrophoresed and treated similarly except that alkali is not used. (First, alkali hydrolyzes RNA, and second, RNA is already single stranded.) For Western blots, proteins are electrophoresed, transferred to nitrocellulose, and probed with a specific antibody.

Southern blotting A technique for detecting the presence of a specific DNA sequence in a genome. The DNA is extracted, cleaved into fragments, separated by gel electrophoresis, denatured, and blotted onto a nitrocellulose filter. There it is incubated under annealing conditions with a radiolabeled probe for the sequence in question, and heteroduplexes of the probe with genomic DNA are detected by radioautography. 

It should be noted that the efficiency of incorporation of phosphate into the CaM kinase II subunits subjected to in situ renaturation is lower than autophosphorylation of the native enzyme in solution (Shackelford and Zivin, 1993). This appeared to be due to immobilization of the enzyme on the membrane and not to the processes of electrophoresis, denaturation, and renaturation. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

The protein was purified by a dialysis procedure, denatured and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting indicated that the protein of interest consisted of two components, one of which increased in concentration as the purification proceeded. The authors initially suggested that this could be due to the presence of a number of species produced by modification of the amino acid side-chains, for example, by glyco-sylation, or by modification of the C- or N- terminus. 

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. 

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

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. 

SDS gel electrophoresis separation in total denaturing conditions was carried out on the protein of culture filtrates and proteins of known molecular mass. The four dark bands (Figure 2) which appear in the gel between 45 and 36 kDa of the standards were assumed to be PG based on the gel filtration results for PG activity and total protein. The relative molecular mass of the four protein bands were estimated as 45 kDa, 42 kDa, 39 kDa and 36 kDa. It was calculated that about 85% of total protein secreted into the culture medium by K. marxianus consisted of PG. 

For CBCA synthase hardly any information has been pubhshed. The enzyme was characterized after it was purified from C. sativa extracts and until this moment no sequence has been deposited. After purification of the protein it was found to be a homodimeric enzyme, meaning that enzyme is formed by two identical domains. This was observed after the purification, when the enzyme had a molecular weight of 136 kDa, and after denatured electrophoresis, when it had a molecular weight of 71 kDa. Furthermore, the CBCA synthase has shown to bear higher affinity for CBGA (1717 M S ) than THCA synthase and CBDA synthase (respectively 1382M s and 1492 M s ), which is probably due to its homodimeric nature [40]. 

G. Muyzer and K. Smalla, Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73 127 (1998). 

T. Vallaeys, E. Topp, G. Muyzer, V. Macheret, G. Laguerre, A. Rigaud, and G. Soulas, Evaluation of denaturing gradient gel electrophoresis in the detection of I6S rDNA sequence variation in rhizobia and methanotrophs. FEMS Microbiol. Ecol. 24 279 (1997). 

G. Muyzer, E. C. de Waal, and A. G. Uitterlinden, Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for I6S rRNA. Appl. Environ. Microbiol. 59 695 (1993). 

G. A. Kowalchuk, J. R. Stephen, W. DeBoer, J. 1. Prosser, T. M. Embley, and J. W. Woldendorp, Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified I6S ribosomal DNA fragments. Appl. Environ. Microbiol. 6.1 1489 (1997). 

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