Glucose Assay demonstration using o-danisidine скачать в хорошем качестве

Glucose Assay demonstration using o-danisidine

This video demonstrates a glucose detection assay using o-danisidine as the chromogenic substrate. I had trouble finding any examples of this assay when I was trying to work out a protocol for running it so thought I would post mine here. The reagents used in this protocol are readily available from Sigma. I used: • o-Dianisidine dihydrochloride (D3252-5G) $69.30 • Peroxidase (P8375-1KU; Sigma) $52.50; 250U/mg, Bottle contains 4mg 20mg/mL – comes as lyophylised powder • Glucose oxidase (G7141-10KU; Sigma) $51.00, 100U/mg; Bottle contains 100mg) – comes as lyophylised powder Making up the Glucose Assay Reagent Step 1: Making up stock o-Dianisidine (5mg/mL) Take the 5g jar of o-Dianisidine dihydrochloride Add: • 0.005g of o-Dianisidine To: • 1mL of RO water Vortex to dissolve Step 2: Making up the Glucose Oxidase Stock Solution (0.8U/µL aliquots) • Take the whole 10,000U vial of glucose oxidase from Sigma • Dissolve the entire vial in 12.5mL RO water • Make this into 10 x 1.25mL aliquots in 1.5mL Eppendorf tubes (Enzyme concentration 0.8U/µL) Snap freeze these aliquots in liquid Nitrogen and store at -20°C until needed. Could freeze dry and store in preproom -20°C freezer (lyophilized enzymes more stable) Step 3: Making up the Assay Reagent Take: • 8.55mL of RO Water (can do it by weight if making at CPC) Add: • 200µL of the 5mg/mL o-Dianisidine dihydrochloride stock solution Add: • One of the 1.25mL aliquot of glucose oxidase (0.8U/µL; 1000U of glucose oxidase) • Use this solution to fully dissolve the whole 1000U bottle of peroxidase Mix Well. This gives 10mL of Assay Reagent which contains: • 0.1mg/mL o-Dianisidine dihydrochloride • 100U/mL glucose oxidase • 100U/mL peroxidase According to the Sigma instructions for the GO Assay Kit (GAGO-20) this made up assay reagent will keep at 2-8°C for 1 month. The Solution should be protected from light, discard if turbidity or colour develops.