5- bromo-4-chloro-3-indolyl-

Aminophthalate anion Atmospheric pressure active nitrogen Analyte pulse perturbation-chemiluminescence spectroscopy Arthromyces rasomus peroxidase Ascorbic acid Adenosine triphosphate Avalanche photodiode 5-Bromo-4-chloro-3-indolyl 2,6-Di-t< r/-bu(yl-4-mclhyl phenol Bioluminescence Polyoxyethylene (23) dodecanol Bovine serum albumin Critical micelle concentration Calf alkaline phosphatase Continuous-addition-of-reagent Continuous-addition-of-reagent chemiluminescence spectroscopy Catecholamines Catechol... 

The arylsulphatase (Ars)-activity in transformants growing in the absence of exogenously supplied arginine was detected qualitatively by screening colonies on plates for their ability to hydrolyze the chromogenic sustrate 5-bromo-4-chloro-3-indolyl-sulphate (X-SO4) according... 

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. 

Colorimetric methods were developed as a nonradioactive alternate method of detection. Enzymes such as HRP or AP, which are usually conjugated to the secondary antibody, can convert substrates to colored precipitates that accumulate on the blot and generate a colored signal. Such substrates include 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for AP and 3,3 -diaminobenzidine... 

List of Abbreviations BCIP, 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt CCD, charged-coupled device CNS, central nervous system cDNA, copy or complimentary RNA cRNA, copy or complimentary RNA DNase, deoxyribonuclease FITC, fluorescein isothiocyanate mRNA, messenger RNA NBT, nitro blue tetrazolium choride PCR, polymerase chain reaction RNase, ribonclease... 

Add the commercially available substrate solution containing 5-Bromo-4-chloro-3-indolyl Phosphate (BCIP)/Nitroblue Tetrazolium (NBT) (SIGMA) to the blot till it is just submerged. 

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. 

The wide range of chromogenic-substrate systems available allows one to obtain excellent color contrast for double/multiple antigen detection. Chromo-genie reactions resulting in black (IGSS), yellow-brown (IPO/DAB), red-brown (IPO/AEC), blue (lAP/Fast Blue, or 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium [BCIP-NBT]), and purple (lAP/Fast Red or New Fuchsin) can be used in different combinations (see Chapter 23). For development of these products, the following protocols are provided (see Note 16). 

BCIP/NBT substrate Dissolve 5-bromo-4-chloro-3-indolyl phosphate at 50 mg/mL in dimethyl formamide. Dissolve nitroblue tetrazolium at 75 mg/mL in 70% (v/v) dimethyl formamide. Add 30 pL of BC1P and 45 pL of NBT to 10 mL of Tris buffer, pH 8.2 immediately before use. 

Bromo-4-chloro-3-indolyl phosphate (BCIP), p-toluidine salt, stock 50 mg/mL... 

The sections are treated for 1 hr with streptavidin-alkaline phosphatase in a humid chamber and then washed in PBS. They are developed for 10 min in nitroblue tetra-zolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). After washing in PBS, the sections are treated for 30 min in a double-staining enhancer (Zymed Laboratories, San Francisco, CA) to prevent the first stain from reacting with the second. They are thoroughly rinsed in distilled water followed by PBS. 

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). 

TK Tn-368 Tn 7 tPA tRNA UTR X-gal YACs virus thymidine kinase cell line originated from the ovary of the insect Trichoplmia ni transposon 7 tissue plasminogen activator transfer ribonucleic acid untranslated regions 5-bromo-4-chloro-3-indolyl- -D-galactoside yeast artificial chromosomes... 

P-galactosidase substrate PBS containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, and 1 mM magnesium chloride, which can be stored at +4°C for approx 2 mo. 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-p-galactopyranoside (X-gal) should be added prior to use. X-gal is made up to 40 mg/mL in dimethyl formamide (DMF) and stored at -20°C in a polypropylene or glass container. 

Prepare the color development solution by adding the BCIP [25 mg of 5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt (Sigma Chemical Co.) in 2.5 mL of N,N -dimethylformamide] and NBT (50 mg of nitro blue tetrazolium in 5 mL of 50% N, N -dimethylformamide) solutions to 100 mL of 1.0 M Tris-HCl, pH 9.0. 

A low detection limit directly influences the sensitivity of the enzyme-based assay. The final enzyme-substrate interaction must yield an ample amount of some end product which can be accurately monitored and, hopefully, quantitated. The authors experiences have been chiefly with enzymatic detection systems which culminate in a visible chromogenic reaction (e.g. alkaline phosphatase, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate). 

Alkaline phosphatase 5-Bromo-4-chloro-3 -indolyl-phosphate with nitroblue tetrazolium chloride (BCIP/NBT) Black/purple... 

Galactosidase 5-Bromo-4-chloro-3-indolyl-/3.D-galactopyranoside (X-Gal) Blue... 

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