Tetrazolium blue, alkaline

Materials Required Silica gel G mobile-phase (1, 2-dichloroethane methanol water 95 5 0.2) 100 ml mixture of chloroform and methanol (9 1) 50 ml solution (1) betamethasone valerate sample 1. 5% w/v solution (2) betamethasone valerate BPCRS/EPCRS 1.5% w/v solution (3) a solution containing 0.030% w/v each of betamethasone EPCRS and betamethasone 21-valerate BPCRS alkaline tetrazolium blue solution q.s. ... 

Procedure Apply separately to the coated TLC plate 1 pi of each of three solutions (1), (2) and (3) prepared in a mixture of chloroform/methanol stated above. After removal of the plate, allow it to cool dry in air until the solvents have evaporated, heat at 105 °C for 10 minutes, cool and spray with alkaline tetrazolium blue solution. 

Reaction of triamcinolone acetonide with tetrazolium blue in alkaline medium gives a blue color (520 mp,) which can be quantitated. It measures the reducing power of the a-ketol-side chain and is useful for general formulation assays. It also is used as a spray reagent in paper and thin layer chromatograms (Section 6.5). 

There are a number of different probes available, including detection probes, which are labelled with a fluorescence marker or, for example, digoxigenin for a further linkage with an antibody-enzyme complex and then a later colorimetric reaction with a chromogenic substrate (such as nitro blue tetrazolium for alkaline phophatase) (Helentjaris McCreery, 1996 Kempf et al., 2000), Capture probes are used to bind the target sequence (RNA or DNA) to a plate or another surface. In most cases the probes are labelled with biotin to react with avidin, which is coated on a plate (Riley, Marshall, Coleman, 1986). 

Evaluation reagents Col-1 ethanolic or methanolic sulfuric acid-l-UV254, Col-2 alkaline tetrazolium blue. Col 3 antimony/III/chloride, Col-4 perchloric acid, Col-5 p-toluene sulfonic acid, Col-6 chloramine T, Col-7 arzenomolybdate, Col-8 phosphomolibdic acid. 

Col-I ethanolic or methanolic sulfuric acid, Col-2 alkaline tetrazolium blue... 

Amin (2001) reported a colorimetric method for vitamin E in pure form and multivitamin capsules based on the reduction of tetrazolium blue to formazan derivative by vitamin E in alkaline medium. The reaction mixture was incubated at 90 2 C for 10 min and the absorbance of the reaction product was monitored at 526 nm with relative standard deviation of 0.7%-1.5%, a limit of detection 0.012 mg/E and sample throughput of approximately 6/h. The reduction of tetrazolium blue to formazan derivative requires 3 h at room temperature and the color developed was stable for 3 h. EDTA was added to sample solution for masking any metal ions during analysis. 

The alkaline phosphatase substrates form precipitates based on either reduction of tetrazolium salts or on the production of colored diazo compounds. Substrates Vector Red, Vector Black, Vector Blue, and BCIP/NBT available from Vector Laboratories produce reaction products which are red, black, blue and purple/blue... 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

Alkaline phosphatase acts on many substrates as well, each precipitating as a different color. For example, a combination of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) results in a permanent blue precipitate at the site of alkaline phosphatase localization. There are other compounds that can also be tried, such as Fast Red TR/Naphthol AS-MX (Sigma, St. Louis, MO), which precipitates as a red color. 

Incubate the filters with 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT). Indoxyl generated from BCIP by the action of alkaline phosphatase condenses to form indigo (blue). Indigo then reacts with NBT to form insoluble diformazan (purple). 

Common chromogen systems currently in use include diaminobenzidine (DAB), 3-amino-9-ethyl-carbazole (AEC), Hanker-Yates reagent, alpha-naphthol pyronin used with peroxidase as substrate fast blue, fast red, BCIP- (5-bromo-4-chloro-3-indolyl phosphate) NBT (nitroblue tetrazolium) used with alkaline phosphatase as substrate tetrazolium, tetranitroblue tetrazolium used with glucose oxidase as substrate, and immunogold with silver enhancement (Leong, 1993 Leong et al, 1997a). 

Oxidation of steroid enones3-Keto-A4-steroids are oxidized by blue tetrazolium (1) in an alkaline medium to 2,6-diketo-A4-steroids in 70-80% yield. Other examples ... 

Wla. Walker, S. W., Howie, A. F., and Smith, A. F., The measurement of glycosylated albumin by reduction of alkaline nitro-blue tetrazolium. Clin. Chim. Acta 156, 197-206... 

BRL DNA Detection kit (no. 530-8239SA). This kit contains 120 jL BRL streptavidm, 60 pL biotinylated calf intestinal alkaline phosphatase, 660 pL nitro-blue tetrazolium (NBT), 500 pL 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 25 pg/pL biotinylated DNA. 

Probes prepared with either digoxigenin- or biotin-modified nucleotides can be hybridized to Southern blots to detect target nucleic acid sequences. These methods offer an attractive alternative to radioactively tagged probes in terms of safety, cost, and efficiency. Most previous nonradioactive strategies utilized the detection of the modified base by the use of a coupled antibody- or avidin-alkaline phosphatase. The blot with the bound alkaline phosphatase was then treated with a compound, such as Nitro Blue Tetrazolium (NBT), that was converted to an insoluble, colored compound at the site of hybridization, thus facilitating visualization of the hybridized probe. 

The visualization of hybridization between the RNA probe and the DNA fragments on the blot is based on an enzyme-linked immunoassay. An antidigoxigenin/alkaline phosphatase conjugate is bound to the digoxigenin component of the probe and then incubated with the substrates X-phosphate and nitroblue tetrazolium (see Section 2.) under alkaline conditions, which will result in purple-blue precipitates on the membrane within a couple of hours. Color detection thus saves time and money as it does not require X-ray films, cassettes, and intensifier screens. For a permanent record the result can be photocopied or photographed. 

Color solution is alkaline phosphatase buffer plus 45 J.L/100mL NBT (75mg/ mL, 4-nitro blue tetrazolium chloride, Roche) and 35 J.L/100mL of BCIP (50mg/mL, 5 Bromo-4-chloro-3-indolyl-phosphate, Roche), and2mM levam-isole (Sigma). Levamisole is made fresh from solid, but may be stored for up to 2wk at4°C. 

---