5-Bromo-4-chloro-3-indolyl phosphate BCIP

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. 

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. 

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

To the second universal add 22 pL of NBT + 17 pL of X-phosphate 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) for both biotin and dig probes. Wrap both universal tubes in tinfoil to keep out the light. 

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. 

APT buffer 100 mM Nad, 100 mM Tiis-Hd, pH9.5,50 mM Mgd, 1% Tween-20. NBT/BCIP staining mix Just before use, add 4.5 pL stock nitro blue tetrazolium salt (NBT) and 3.5 pL stock 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 1 mL APT buffer containing 2 mM levamisole. Stock NBT is 75 mg/mL in 70% dimethyl formamide, and stock BCIP is 50 mg/mL in 100% dimethyl formamide both stocks are supplied by Gibco-BRL as Immunoselect kit. 

In the case of the mosses investigated by Turner et al. (2001), the azo-dye bromo-4-chloro-3-indolyl phosphate (BCIP), which forms a blue/purple colour following hydrolysis (Coston and Holt, 1958), proved to be the most useful stain. Staining occurred in different locations in different species. In some species (e.g. Hylocomium splendens and Racomitrium lanuginosum] staining occurred inside the cell wall. 

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... 

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. 

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). 

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. 

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

Concentrated substrate solution (NBT/BCIP Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in dimethylformamide, to be diluted 1 100 in substrate buffer before use). 

BCIP (= X-phosphate 5-bromo-4-chloro-3-indolyl phosphate) 1.75 mg is dissolved in 35 pi of 100% DMF and added dropwise with gentle mixing into the NBT solution. Final concentration 0.33 mg/ml NBT and 0.17 mg/ ml BCIP. 

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. 

---