GlucoseFluorescent Detection Kit (Two Plate)

Catalog No. ABIN2815080

Product Details

Target: Glucose

Detection Method: Fluorometric

Host: Human

Application: Detection (D)

Purpose: The DetectX® Glucose Fluorescent Detection Kit is designed to quantitatively measure glucose in a variety of samples.

Brand: DetectX®

Sample Type: Biological Buffers, Cell Culture Supernatant, Plasma, Serum, Urine

Components: Black 96 well Half Area Plates 2 Plates Corning Costar Plate 3694.
Glucose Standard 90 μL Glucose at 100 mg/dL in a special stabilizing solution.
Assay Buffer 50 mL Assay buffer containing detergents and stabilizers.
Substrate 5 mL A solution of the substrate in a special stabilizing buffer.
Horseradish Peroxidase Concentrate 60 μL A 100X concentrated solution of HRP in a special stabilizing solution.
Glucose Oxidase Concentrate 600 μL A 10X concentrated solution of Glucose Oxidase in a special stabilizing solution.

Material not included: Repeater pipet with disposable tips capable of dispensing 25 μL. 96 well plate reader capable of reading fluorescence at 580-590 nm with excitation at 570-580 nm.
Set plate parameters for a 96-well Corning Costar 3694 plate.
See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
Software for converting colorimetric intensity readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Protocol: A ß-D- glucose standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
Samples are mixed with the Substrate and horseradish peroxidase and the reaction initiated by addition of glucose oxidase.
The reaction is incubated at room tem- perature for 30 minutes.
The glucose oxidase reacts with glucose to produce hydrogen peroxide which, in the presence of HRP, converts the substrate into a fluorescent product.
The fluorescent product is read at 590 nm with excitation at 570 nm.
Increasing levels of glucose cause a linear increase in fluorescence.

Reagent Preparation:

Horseradish Peroxidase (HRP) and Glucose Oxidase (GOD) Preparation Dilute the HRP Stock solution 1:100 with Assay Buffer using the table below: HRP Dilution Table 1/2 Plate One Plate Two Plates HRP Stock 15 μL 28 μL 55 μL Assay Buffer 1.485 mL 2.772 mL 5.445 mL Total Volume 1.5 mL 2.8 mL 5.5 mL Dilute the GOD Stock solution 1:10 with Assay Buffer using the table below: GOD Dilution Table 1/2 Plate One Plate Two Plates GOD Stock 150 μL 275 μL 550 μL Assay Buffer 1.350 mL 2.475 mL 4.95 mL Total Volume 1.5 mL 2.75 mL 5.5 mL

Sample Preparation:

Samples that need to be stored after collection should be stored at -70 °C or lower,preferably after being frozen in liquid nitrogen.

Assay Procedure:

Use the plate layout sheet on the back page to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3694 plate.
1. Pipet 20 μL of diluted samples or standards into duplicate wells in the plate.
2. Pipet 20 μL of Assay Buffer into duplicate wells as the Zero standard.
3. Add 25 μL of the prepared HRP solution to each well using a repeater pipet.
4. Add 25 μL of the Colorimetric Substrate solution to each well using a repeater pipet.
5. Initiate the reaction by adding 25 μL of the prepared GOD solution to each well using a repeater pipet.
6. Incubate at room temperature for 30 minutes.
7. Read the fluorescent emission at 585 ± 5 nm with excitation at 575 ± 5 nm. Please contact your plate reader manufacturer for suitable filter sets.

Calculation of Results:

Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit, after subtracting the mean FLU for the Zero wells.
The sample concentrations obtained should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from: http://www.myassays.com/arbor-assays-glucose-fluorescent-detec- tion-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean FLU Net FLU Glucose Conc. (mg/dL) Zero 867 0 0 Standard 1 37,595 36,728 10 Standard 2 30,806 29,939 5 Standard 3 21,167 20,300 2.5 Standard 4 13,197 12,330 1.25 Standard 5 7,449 6,582 0.625 Standard 6 4,284 3,416 0.313 Standard 7 2,572 1,705 0.156 Sample 1 29,137 28,269 4.47 Sample 2 7,153 6,285 0.602 Always run your own standard curves for calculation of results.
Do not use these data.
Conversion Factor: 100 mg/dL of Glucose is equivalent to 1 mg/mL or 5.51 mM.

Assay Precision: Three diluted human serum samples were run in replicates of 20 in an assay.
Inter Assay Precision:
Three diluted human serum samples were run in duplicate in twelve assays run over multiple days by three operators.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
This product is not for Human Diagnostic Use.

Handling Advice: SaMple typeS and preparatiOn Samples that need to be stored after collection should be stored at -70°C or lower, preferably af- ter being frozen in liquid nitrogen.
Serum and plasma samples can be used after being diluted ≥ 1:15.
Urine samples can be used after being diluted ≥ 1:2.
This assay has been validated for serum, plasma, urine, buffer and media samples.

Storage: 4 °C,RT

Storage Comment: All components of this kit should be stored at 4°C until the expiration date of the kit.

Target Details

Target: Glucose

Background: Glucose (C6H12O6) is by far the most common carbohydrate. It is a monosaccharide, an aldose, a hexose, and a reducing sugar and is also known as dextrose, because it is dextrorotatory (rotates polarized light clockwise). The structure of glucose is shown below as both the straight chain and cyclic forms. Glucose Structures For all biological and molecular events and for multiple cellular functions, energy is essential. En- ergy is available in the form of ATP (adenosine triphosphate), most of which is generated through aerobic cellular respiration of carbohydrate and glucose, the major source of biological free energy in higher organisms. Reduced energy levels threaten cellular homeostasis and integrity. Impaired energy metabolism may trigger pro-apoptotic signaling (programmed cell death), oxidative dam- age, excitotoxicity and impede mitochondrial DNA repair1. A serious fall in blood glucose can be characterized by metabolic dysfunction, neuroglycopenia, seizure, and death2. A persistent elevation in blood glucose leads to "glucose toxicity." Glucose toxicity contributes to ß-cell dysfunction and the pathology grouped together as complications of diabetes. Estrogen-induced signaling pathways in hippocampal and cortical neurons involve the mitochondria to enhance mitochondrial function and to sustain aerobic glycolysis and citric acid cycle oxidative phosphorylation and ATP generation