Assays electrophoresis

In this chapter, the dimethylenblue method as a urinary screening assay, electrophoresis, and TLC for GAG differentiation and the enzyme assays will be introduced. Furthermore, pitfalls will be discussed and a short survey of alternative methods will be given. 

J. Wang, On-chip enzymatic assays, Electrophoresis, 23 (2002) 713-718. J. Wang and M.P. Chatrathi, Microfabricated electrophoresis chip for bioassay of renal markers, Anal. Chem., 75 (2003) 525-529. 

Fried MG (1989) Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis 10(5-6) 366-376. doi 10.1002/elps.ll50100515... 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

The developed assay was successfully applied for the arsenite and arsenate determination in contaminated waters of the gold recovery plant and in snow covers of the industrial anthropogenic sources vicinities as well. The data produced are in a good agreement with the results of independent methods atomic absorptioin and atomic emission spectrometry and capillary electrophoresis. 

Gasiorowski, K., Brocos, B. DNA repair of hydrogen peroxide-induced damage in human lymphocytes in the presence of four antimutagens. A study with alkaline single cell gel electrophoresis (comet assay). Cellular Molecular Biology Letters, Vol.6, (2001), pp. 897-911, ISSN 1425-8153... 

The isoenzymes can be separated by electrophoresis on cellulose acetate, and Roberts, et al ( ) have described a method whereby the separated isoenzymes are eluted and then assayed kinetically. 

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). 

Rosalki, S. B. Standardization of isoenzyme assays with special reference to lactate dehydrogenase isoenzyme electrophoresis. Clin. Biochem. (1974), 2> 29-40. 

SDS-PAGE was performed by the method of Laemmli [17]. The methods for native PAGE, isoelectric focussing, detection of esterase activity in electrophoresis gels, and assays for protein glycosylation have been described elsewhere [5]. 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

Cann, J.R., Models of mobility-shift assay of complexes between dimerizing protein and DNA, Electrophoresis, 18, 1092, 1997. 

Cann, J.R., Theoretical studies on the mobility-shift assay of protein-DNA complexes, Electrophoresis, 19, 127, 1998. 

Enzyme-Linked Immunoadsorbent Assay (NRL) Multiplexed Sandwich Fluoroimmunoassay (LLNL) Capillary Electrophoresis (LLNL)... 

In a different approach a super-high-throughput ee-assay was developed on the basis of chirally modified capillary array electrophoresis (CAE).90 CAE was used in the Human Genome Project, and commercially available instruments have been developed which comprise a high number of capillaries in parallel, for example the 96-capillary unit MegaBACE consisting of 6 bundles of 16 capillaries.91 The system can address a 96-well microtiter plate. It was adapted to perform ee-determinations of chiral amines, which are potentially accessible by catalytic reductive amination of ketones, transition metal catalyzed Markovnikov addition of ammonia, or enzymatic hydrolysis of acetamides (Scheme 14).90... 

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