| Description | BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/ Nitro Blue Tetrazolium) liquid ready-to-use substrate is a highly active and stable formulation utilized for colorimetric detection of Alkaline Phosphatase (AP) activity in membrane assays. Positive reactions form an intense blue/purple precipitate at BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/ Nitro Blue Tetrazolium) liquid ready-to-use substrate is a highly active and stable formulation utilized for colorimetric detection of Alkaline Phosphatase (AP) activity in membrane assays. Positive reactions form an intense blue/purple precipitate at the site of the reaction. The color develops when AP catalyzes the dephosphorylation of BCIP and converts NBT to insoluble blue/purple NBT formazan. The intense blue/purple precipitate is very stable and resists fading when exposed to light.Product Characteristics BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/ Nitro Blue Tetrazolium) liquid, ready-to-use substrate is a highly active and stable formulation utilized for colorimetric detection of Alkaline Phosphatase (AP) activity in membrane assays. Positive reactions form an intense blue/purple precipitate at the site of the reaction. The color develops when AP catalyzes the dephosphorylation of BCIP and converts NBT to insoluble blue/purple NBT formazan. The intense blue/purple precipitate is very stable and resists fading when exposed to light.Composition & Properties The BCIP/NBT is a liquid, ready-to-use substrate. The solution contains NBT, BCIP and non-toxic stabilizers in an aqueous buffer, pH 9.6. The formulation is free of organic solvents.Working Procedure 1.Allow the solution to reach room temperature prior to use. 2.After the final incubation with the AP-labelled probe, wash the membrane thoroughly in a Tris-Buffered Saline (TBS Buffer) containing 0.1% Tween 20. 3.After the final wash, completely cover the membrane with BCIP/NBT solution and allow the color reaction to develop until optimal signal intensity is reached (usually 5-30 minutes). Incubation time will depend on enzyme activity. A further adjustment of the dilution of the AP probe may be necessary. The formazan deposit may flake off the membrane or background staining of the membrane may occur if the color development is too fast or too slow, respectively. 4.Stop the enzyme reaction by thoroughly washing membrane with deionized water. 5.Dry the membrane and store at room temperature. It is recommended to protect the membrane from light for a permanent record of results.Tips & Tricks Normal appearance of the solution is clear, pale yellow. Discard if solution is turbid or purple. Crystalline precipitate can occur in the product. Do not use phosphate buffers, as inorganic phosphate is a powerful inhibitor of AP. The products are not recommended for immunohistochemical or in situ hybridization techniques.Handling & Storage Store solution at 2-8 °C in the dark. Avoid exposure to light and heat. Re-dispense only into bottles made of High Density Polyethylene (HDPE), amber color. Dispensing guidelines are available upon request... Read More | Inquire | Inquire | Acid phosphatase is an esterase with broad activity at an optimal pH below 7.0. There are three isozymes, EI, EII, and EIII of similar molecular weight (55 kDa± 5 kDa). Their optimum pH's are 5.5, 4.5, and 4.0 respectively. Acid phosphatase activity was observed by Teller Aladdin Library Acid phosphatase is an esterase with broad activity at an optimal pH below 7.0. There are three isozymes, EI, EII, and EIII of similar molecular weight (55 kDa± 5 kDa). Their optimum pH's are 5.5, 4.5, and 4.0 respectively. Acid phosphatase activity was observed by Teller Aladdin Library Archives in 1954 in preparations of a wheat germ lipase described by Singer JBC, 174, 11, in 1948. Equivalent commercial preparations have been distributed labeled as lipase and acid phosphatase thus generating some confusion. Subsequent work has confirmed that the non-specific esterase activity of the wheat germ preparation may be measured both as lipase (triacetin as substrate) and phosphatase. The enzyme assay is based on the work of Brandenberger and Hanson (Helv. Chim. Acta, 36, 900, 1953) and Hofstee ( Arch. Biochem. Biophys., 51, 239, 1954).Acid phosphatase (APase) non-specifically catalyzes the hydrolysis of monoesters and anhydrides of phosphoric acid to produce inorganic phosphate. It is used to study the production, transport, and recycling of phosphate and the metabolic and energy transduction processes of the cell.Characteristics of Acid Phosphatase from Wheat Germ:Molecular weight: 55,000 ± 5,000 (Verjee 1969).Composition: Three isozymes of closely similar molecular weights have been reported by Verjee (1969): EI, EII, and EIII. See also Brouillard and Ouellet (1965).Optimal pH: EI - 5.5, EII - 4.5, and EIII - 4.0. (Verjee 1969).Specificity: The enzyme has a broad esterase activity. See Joyce and Grisolia (1960). It shows highest activity for pyrophosphate.Inhibitors: Fluoride, molybdate and orthophosphate (Verjee 1969)... Read More | Purity≥95% SDS-PAGE. Recombinant human MIF, fused to His-tag at N-terminus, was cloned into an E. coli expression vector and was purified to apparent homogeneity by using conventional column chromatography techniques.FunctionPro-inflammatory cytokine. Involved in the innate immune response to Purity≥95% SDS-PAGE. Recombinant human MIF, fused to His-tag at N-terminus, was cloned into an E. coli expression vector and was purified to apparent homogeneity by using conventional column chromatography techniques.FunctionPro-inflammatory cytokine. Involved in the innate immune response to bacterial pathogens. The expression of MIF at sites of inflammation suggests a role as mediator in regulating the function of macrophages in host defense. Counteracts the anti-inflammatory activity of glucocorticoids. Has phenylpyruvate tautomerase and dopachrome tautomerase activity (in vitro), but the physiological substrate is not known. It is not clear whether the tautomerase activity has any physiological relevance, and whether it is important for cytokine activity... Read More |