Galactose Colorimetric Detection Kit

Produktnummer ABIN2815084

Produktdetails

Target: Galactose

Nachweismethode: Colorimetric

Applikation: Detection (D)

Verwendungszweck: The DetectX® Galactose Colorimetric Detection Kit is designed to quantitatively measure galactose in a variety of samples.

Marke: DetectX®

Proben: Biological Buffers, Cell Culture Supernatant, Plasma, Serum

Bestandteile: Clear 96 well Half Area Plates 2 Plates Corning Costar Plate 3695.
Galactose Standard 90 μL Galactose at 250 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.
Galactose Oxidase 2 Vials Freeze dried solution of Galactose Oxidase stored in a desiccator.

Benötigtes Material: Repeater pipet with disposable tips capable of dispensing 25 μL. 96 well plate reader capable of reading at 560 nm (Acceptable Range 540-580 nm.).
Set plate parameters for a 96-well Corning Costar 3695 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.

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Protokoll: A D-(+)-galactose 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 Colorimetric Substrate and horseradish peroxidase and the reaction initiated by addition of galactose oxidase.
The reaction is incubated at room temperature for 30 minutes.
The galactose oxidase reacts with galactose to produce hydrogen peroxide which, in the presence of HRP, reacts with the colorless Colorimetric Substrate to produce a pink-colored product which is read at 560 nm.
Increasing levels of galac- tose cause a linear increase in color.

Aufbereitung der Reagenzien:

Horseradish Peroxidase (HRP) 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 30 μL 55 μL Assay Buffer 1.485 mL 2.97 mL 5.445 mL Total Volume 1.5 mL 3 mL 5.5 mL Galactose Oxidase (GOD) Preparation Allow the desiccator to warm to room temperature.
Add 3.125 mL of the Assay Buffer to a Galactose Oxidase vial and vortex thoroughly.
Each vial contains enough GOD for one plate.
Unused prepared Galactose Oxidase solution should be stored at -20 °C after reconstitution.

Testdurchführung:

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 3695 plate. See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
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.
4. Initiate the reaction by adding 25 μL of the prepared GOD solution to each well using a repeater pipet.
5. Incubate at room temperature for 30 minutes.
6. Read the plate at 560 nm (Acceptable Range 540-580 nm.).

Ergebnisberechnung:

Average the duplicate OD 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 ODs for the Zero wells.
The sample concentrations ob- tained should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from www.myassays.com/arbor-assays-galactose-colorimetric-detection- kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean OD Net OD Galactose Conc. (mg/dL) Zero 0.099 0.000 0 Standard 1 1.623 1.524 25 Standard 2 1.374 1.275 12.5 Standard 3 0.830 0.731 6.25 Standard 4 0.494 0.395 3.125 Standard 5 0.267 0.168 1.56 Standard 6 0.187 0.088 0.781 Sample 1 1.122 1.023 9.04 Sample 2 0.454 0.355 2.97 Always run your own standard curves for calculation of results.
Do not use these data.
Conversion Factor: 100 mg/dL of Galactose is equivalent to 1 mg/mL or 5.55 mM.

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

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Vorsichtsmaßnahmen: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
This product is not for Human Diagnostic Use.
Sample typeS and preparatiOn Samples that need to be stored after collection should be stored at -70°C or lower, preferably after being frozen in liquid nitrogen.
Serum and plasma samples can be used after being diluted ≥ 1:15.
This assay has been validated for buffer and media samples.

Lagerung: -20 °C,4 °C,RT

Informationen zur Lagerung: All components of this kit should be stored at 4°C until the expiration date of the kit. Once reconstituted, the Galactose Oxidase must be stored at -20°C.

Antigendetails

Target: Galactose

Hintergrund: Galactose is a hexose sugar that differs from glucose only by the configuration of the hydroxyl group at the carbon-4 position. Present as an anomeric mixture of α-D-galactose and ß-D-galac- tose, this monosaccharide exists abundantly in milk, dairy products and many other food types such as fruits and vegetables1,2. Absorption of galactose in humans is mediated by the Na+/glu- cose co-transporters SGLT1 and SGLT2 from food across the brush border membrane of the proxi- mal jejunum and renal epithelium3-6. Other sources of galactose include endogenous production and natural turnover of glycolipids and glycoproteins. Adult humans can produce up to 2 grams of galactose per day7. Galactose Inside the cells, ß-D-galactose is epimerized to α-D-galactose through the action of a mutarotase8. α-D-galactose is subsequently converted to galactose-1-phosphate (Gal-1-P) by the enzyme ga- lactokinase. In the presence of galactose-1-phosphate uridylyltransferase, Gal-1-P reacts with UDP-glucose to form UDP-galactose and glucose-1-phosphate. Glucose-1-phosphate produced can enter the glycolytic pathway or react with UTP in the presence of UDP-glucose pyrophospho- rylase to form a new molecule of UDP-glucose. This enzyme pathway comprises the evolution- arily conserved Leloir pathway of galactose metabolism. If the flow of galactose through the Leloir pathway is perturbed either due to congenital deficiency of any of the above-mentioned enzymes or an overwhelming presence of this hexose, toxicity syndromes (galactosemia) will be observed. Its cause as a defect in galactose metabolism was identified by a group led by Kalckar in 19569