Glucose Assay Kit, BioAssay™, Colorimetric/Fluorometric

Glucose (C6H12O6) is a key diagnostic parameter for many metabolic disorders. Increased glucose levels have been associated with diabetes mellitus, hyperactivity of thyroid, pituitary and adrenal glands. Decreased levels are found in insulin secreting tumors, myxedema, hypopituitarism and hypoadrenalism. Simple, direct and high-throughput assays for measuring glucose concentrations find wide applications in research and drug discovery. Glucose assay kit uses a single Working Reagent that combines the glucose oxidase reaction and color reaction in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to glucose concentration in the sample.

Sensitive and accurate. Use as little as 20ul samples. Linear detection range in 96-well plate: 5 to 300uM (90 ug/dL to 5.4 mg/dL) glucose for Colorimetric Assay:s and 1 to 30uM for Fluorometric Assay:s.
Simple and high-throughput. The procedure involves addition of a single working reagent and incubation for 30 min at room temperature.

Direct Assays: glucose in serum, plasma, urine, saliva, milk, culture medium and other biological samples.
Drug Discovery/Pharmacology: effects of drugs on glucose metabolism.
Food and Beverages: glucose in food, beverages etc.

Assay Buffer: 10ml Enzyme Mix: 120ul
Dye Reagent: 120ul Standard: 1ml 300 mg/dL Glucose
Storage conditions. The kit is shipped on dry ice. Store Assay Buffer at 4°C and other components at -20°C. Shelf life: 6 months after receipt.
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Sample treatment: saliva samples should be centrifuged for 5 min at 14,000 rpm prior to assay. Milk samples should be cleared by mixing 100ul 6N HCl and 600ul milk. Centrifuge 5 min at 14,000 rpm and transfer supernatant into a clean tube. Add 170ul 6N NaOH perml supernatant. Mix well and centrifuge again at 14,000 rpm. The supernatant can be assayed. The dilution factor in this procedure is n=1.36. Samples can be analyzed immediately after collection, or stored in aliquots at –20 °C. Avoid repeated freeze-thaw cycles. If particulates are present, centrifuge sample and use clear supernatant for assay.
1. Equilibrate all components to room temperature. During experiment, keep thawed Enzyme in a refrigerator or on ice.
2. Standards and samples: for 300uM standard, mix 15ul 300 mg/dL standard with 818ul dH2O. Dilute standard in dH2O as follows.
No 300uM STD+H2O Vol (ul) Glucose (uM)
1 200ul+0ul 200 300
2 120ul+80ul 200 180
3 60ul+140ul 200 90
4 0ul+200ul 200 0
Transfer 20ul standards and samples into separate wells.
3. Working Reagent. For each reaction well, mix 85ul Assay Buffer, 1ul Enzyme Mix (vortex briefly before pipetting), and 1ul Dye Reagent in a clean tube. Transfer 80ul Working Reagent into each reaction well. Tap plate to mix.
4. Incubate 30 min at room temperature. Read optical density at 570nm (550-585nm).

Subtract blank OD (water, #4) from the standard OD values and plot the DOD against standard concentrations. Determine the slope and calculate the glucose concentration of Sample, [Glucose] =
ODSAMPLE–ODBLANK
Slope
(uM)
ODSAMPLE, ODBLANK are optical density values of the sample and water.
Conversions: 1 mg/dL glucose equals 55.5uM, 0.001% or 10 ppm.

For Fluorometric Assays, the linear detection range is 1 to 30uM glucose. Mix 10ul of the standards from Colorimetric Procedure: with 190ul dH2O to obtain standards at 30, 18, 9, 0uM glucose. Transfer 20ul standards and 20ul samples into separate wells of a black 96-well plate. Add 80ul Working Reagent (see Colorimetric Procedure:), tap plate to mix. Incubate 30 min at room temperature and read fluorescence at lex=530nm and lem=585nm. The glucose concentration of Sample is calculated as
[Glucose]=(uM)
FSAMPLE-FBLANK
Slope
Notes: (1). If the calculated sample glucose concentration is higher than 300uM in Colorimetric Assay: or 30uM in Fluorometric Assay:, dilute sample in water and repeat the assay. Multiply result by the dilution factor. (2). To determine glucose in phenol red culture medium, dilute both sample and glucose standards in the same glucose free medium for Colorimetric Assay:. For Fluorometric Assay:, prepare standards in phenol red medium. Dilute sample and standards 20-fold or more in water.

Pipetting devices, centrifuge tubes, clear flat-bottom 96-well plates, black 96-well plates and plate reader.

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.