Signaling Pathway Assays
Succinate Assay Kit (BA0149)
- SKU:
- BA0149
- Product Type:
- Assay
- Instrument:
- Microplate Reader
- Sample Type:
- Cells, Tissue
- Research Area:
- TCA Cycle
- Research Area:
- Diabetes & Obesity
- Research Area:
- Food Safety & Analysis
- Research Area:
- Plant & Environmental Stress
- Research Area:
- Clinical Chemistry
Description
Succinate Assay Kit (BA0149) (BA0149)
The Succinate Assay Kit (SKU: BA0149) provides a simple, one-step method for measuring succinate (succinic acid) in food, beverage, agricultural and other biological samples. Succinate is found in all plant and animal tissues, is an intermediate in the citric acid cycle and plays an important role in intracellular energy generation. In this assay succinate is converted to pyruvate, which reacts with specific reagents and a dye to form a coloured product. The colour intensity at 570 nm, or the fluorescence intensity at 530/585 nm, is directly proportional to the succinate concentration in the sample. The room-temperature format requires no 37 C heater, uses 20 uL of sample and is readily automated for high-throughput use.
| Product Name: | Succinate Assay Kit (BA0149) |
| SKU: | BA0149 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 10 - 400 uM; fluorometric 2 - 40 uM succinate |
| Sample Type: | Food, beverage, agricultural products and other biological 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 one-step enzymatic assay for the quantitative colorimetric or fluorometric determination of succinate. Succinate is converted to pyruvate, which reacts with a dye to form a coloured product at 570 nm or a fluorescent product at 530/585 nm. The room-temperature procedure adds a single working reagent and reads after 30 min.
- Fast and sensitive, using 20 uL of sample
- Linear detection range: colorimetric 10 - 400 uM, fluorometric 2 - 40 uM succinate
- Convenient room-temperature assay requiring no 37 C heater
- High-throughput 96-well plate format
- Direct measurement of succinate in food, beverage, agricultural products and other biological 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 PEP by adding 120 uL water and mixing thoroughly. Store reconstituted PEP at -20 C and use within one month. |
| 2 | Prepare samples: assay clear and slightly coloured samples directly; homogenise solid samples in water and filter or centrifuge (5 min at 14,000 rpm). |
| 3 | Colorimetric assay uses an internal standard. Add 20 uL of each sample to two wells and 20 uL water to a further well. Prepare 1 mM succinate standard (20 uL of 20 mM in 380 uL water); add 5 uL to the sample-plus-standard wells and 5 uL water to the sample-alone and water wells. |
| 4 | Prepare Working Reagent per well by mixing 85 uL Assay Buffer, 1 uL Enzyme Mix, 1 uL Cosubstrate, 1 uL PEP and 1 uL Dye Reagent. Add 80 uL to each well, tap to mix and incubate for 30 min at room temperature. |
| 5 | Read optical density at 570 nm (550-585 nm). |
| 6 | For the fluorometric assay, prepare 40, 24, 12 and 0 uM standards, transfer 20 uL standards and samples into a black plate, add 80 uL Working Reagent and read fluorescence at 530/585 nm after 30 min. |
Colorimetric: [Succinate] = ((R_SAMPLE - R_WATER) / (R_STANDARD - R_SAMPLE)) x 250 x n (uM), where R values are the optical densities of the sample, water and sample-plus-standard, and n is the dilution factor. Fluorometric: determine the slope from the standards and calculate [Succinate] = ((R_SAMPLE - R_WATER) / Slope) x n (uM). One mM succinate equals 11.7 mg/dL or 117 ppm.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| Enzyme Mix | 120 uL | -20 C |
| Cosubstrate | 120 uL | -20 C |
| PEP | Dried | -20 C |
| Dye Reagent | 120 uL | -20 C |
| Standard (20 mM Succinate) | 500 uL | -20 C |
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