| 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 | Product DescriptionEndo F2 cleaves N-linked (asparagine-linked) biantennary oligosaccharides from glycoproteins. It also will cleave high mannose glycans but at a 40x reduced rate. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, Product DescriptionEndo F2 cleaves N-linked (asparagine-linked) biantennary oligosaccharides from glycoproteins. It also will cleave high mannose glycans but at a 40x reduced rate. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.Endoglycosidase F2 is less sensitive to protein conformation than PNGase F and is therefore more suitable for deglycosylation of native proteins. However, for optimal results, denaturation of the glycoprotein is recommended.Contents60 µl aliquot of enzyme (0.3 U) in 10 mM sodium acetate 25mM NaCl, pH 4.5Included with 20 µL and 60 µL pack sizes:5x Reaction Buffer – 250 mM sodium acetate, pH 4.5Molecular weight 32,000 daltonsSpecific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured porcine fibrinogen in 1 minute at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved fibrinogen migrates faster).Formulation The enzyme is provided as a sterile-filtered solution in 10 mM sodium acetate, 25mM NaCl, pH 4.5Specificity Endo F2 cleaves Asparagine-linked biantennary and high mannose glycans (at a 40X reduced rate). It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Endoglycosidase F2 is less sensitive to protein conformation than PNGase F and is therefore more suitable for deglycosylation of native proteins. However for optimal results, denaturation of the glycoprotein is recommended.Quality & Purity Endo F2 is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer 4.5 3. Add 2.0 µl of Endo F2 to the reaction. Incubate 1 hour at 37°C. Monitor cleavage by SDS-PAGEThe production host strain has been extensively tested and does not produce any detectable glycosidases... Read More | Inquire | Inquire | Inquire |