Description
Isocitrate Assay Kit (Fluorometric) (BA0103) (BA0103)
The Isocitrate Assay Kit (Fluorometric) (SKU: BA0103) measures isocitrate through the NADPH generated when isocitrate is oxidised by isocitrate dehydrogenase. Isocitrate is a substrate in the citric acid (TCA) cycle and is commonly found in fruits and vegetables, where it is used industrially as a marker of juice quality and purity. In this assay the NADPH reduces a probe to a highly fluorescent product, the intensity of which, measured at 530/585 nm, is proportional to the isocitrate concentration. The homogeneous mix-incubate-measure format is fast, sensitive and readily automated for high-throughput screening.
| Product Name: | Isocitrate Assay Kit (Fluorometric) (BA0103) |
| SKU: | BA0103 |
| Detection Method: | Fluorometric (530/585 nm) |
| Detection Range: | 0.6 - 500 uM (20 uL sample, 10 min reaction) |
| Sample Type: | Food, beverage, cell lysate, tissue homogenate, serum and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 10 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A rapid, sensitive fluorometric assay for isocitrate suitable for food, beverage and biological samples, requiring only a 20 uL sample and a 10-minute reaction.
- Fast and sensitive, with a linear detection range of 0.6 - 500 uM for a 10-minute reaction
- Convenient, high-throughput homogeneous mix-incubate-measure format
- Readily automated for high-throughput screening
- Isocitrate determination in food, beverage and biological samples such as cell lysate, tissue homogenate and serum
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 | Sample preparation: rinse tissue in PBS, homogenise (50 mg) in ~200 uL 50 mM potassium phosphate (pH 7.5) and centrifuge at 10,000 g for 15 min at 4C. For cells, collect by centrifugation, homogenise or sonicate in cold 50 mM potassium phosphate (pH 7.5) and centrifuge at 14,000 g for 10 min at 4C. Use supernatant for the assay. |
| 2 | Keep thawed Enzyme A and B on ice; equilibrate other reagents to 25C and centrifuge tubes briefly. |
| 3 | Standards: prepare 1000 uL of 500 uM Premix by mixing 5 uL of the 100 mM Standard with 995 uL distilled water, then dilute per the table. Transfer 20 uL of each standard into a black flat-bottom 96-well plate. |
| 4 | Transfer 20 uL of each sample into separate wells. |
| 5 | Prepare Working Reagent per well from 4 uL Probe, 4 uL NADP, 1 uL Enzyme A, 1 uL Enzyme B and 75 uL Assay Buffer. Add 80 uL to each sample well and tap to mix. |
| 6 | Incubate at room temperature for 10 minutes and read fluorescence at 530/585 nm. |
Subtract the water blank from the standard values and plot delta-F against standard concentration. [Isocitrate] = (F_SAMPLE - F_BLANK) / Slope x n (uM), where n is the sample dilution factor. If the calculated concentration exceeds 500 uM, dilute in water and repeat. 1 uM is equivalent to 189 ug/L or 0.189 ppm isocitrate.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| NADP | 500 uL | -20C |
| Standard | 1 mL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| Probe | 750 uL | -20C |
