Description
D-Sorbitol Assay Kit (Colorimetric) (BA0147) (BA0147)
The D-Sorbitol Assay Kit (SKU: BA0147) provides a fast and sensitive colorimetric method for measuring sorbitol in biological, food, beverage and agriculture samples. Sorbitol (glucitol) is a sugar alcohol obtained from glucose by reducing an aldehyde group to a hydroxyl group, and its accumulation in erythrocytes, retinal cells and Schwann cells has been associated with retinopathy, cataracts, peripheral neuropathy and diabetes. It is widely used as a sugar substitute and as a laxative. This assay involves an end-point enzyme-coupled MTT/NAD reaction that forms a coloured product with an absorption maximum at 565 nm. The increase in absorbance at 565 nm is directly proportional to the sorbitol concentration. The room-temperature format requires no 37 C incubator and is readily automated for high-throughput use.
| Product Name: | D-Sorbitol Assay Kit (Colorimetric) (BA0147) |
| SKU: | BA0147 |
| Detection Method: | Colorimetric |
| Detection Range: | 5 - 1000 uM D-sorbitol |
| Sample Type: | Biological (e.g. blood), food, beverage and agriculture samples |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20 C upon receipt. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A colorimetric assay for the quantitative determination of D-sorbitol. An end-point enzyme-coupled MTT/NAD reaction forms a coloured product measured at 565 nm, with absorbance proportional to sorbitol concentration. The homogeneous mix-incubate-measure format is performed at room temperature and is suitable for high-throughput screening.
- Fast and sensitive, with a linear detection range of 5 - 1000 uM D-sorbitol (20 uL sample)
- Room-temperature assay requiring no 37 C incubator
- Convenient homogeneous mix-incubate-measure format
- Readily automated on HTS liquid-handling systems
- Sorbitol determination in biological (e.g. blood), food, beverage and agriculture samples
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Reconstitute Enzyme A by adding 120 uL distilled water and mixing thoroughly (stable at least one week at -20 C). |
| 2 | Prepare samples: extract sorbitol in water; homogenise solid samples and filter or centrifuge (5 min at 14,000 rpm). Adjust sample pH to 7-8 if required, and deproteinate serum or plasma using 10 kDa membrane filters. |
| 3 | Prepare standards: make a 1000 uM premix (10 uL of 50 mM Standard in 490 uL water) and dilute to 1000, 600, 300 and 0 uM. Transfer 20 uL of each standard into wells of a clear flat-bottom 96-well plate. |
| 4 | Transfer 20 uL of each sample in duplicate; for visibly coloured samples, include a Sample Blank. |
| 5 | Prepare Working Reagent per well by mixing 75 uL Assay Buffer, 1 uL Enzyme A, 1 uL Enzyme B and 8 uL NAD/MTT. For Sample Blanks, prepare a blank reagent without Enzyme A. |
| 6 | Add 80 uL of the appropriate Working Reagent to each well, tap to mix and incubate for 30 min at room temperature. |
| 7 | Read optical density at 565 nm (520-600 nm). |
Subtract the water blank from the standard values and plot the change in optical density against sorbitol concentration to determine the slope. Calculate [Sorbitol] = ((ODSAMPLE - ODBLANK) / Slope) x n (uM), where n is the dilution factor. If the concentration exceeds 1000 uM, dilute the sample in water and repeat. One mM sorbitol equals 18.2 mg/dL, 0.018% or 182 ppm.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| NAD/MTT | 1 mL | -20 C |
| Enzyme A | Dried | -20 C |
| Enzyme B | 120 uL | -20 C |
| Standard (50 mM Sorbitol) | 250 uL | -20 C |
