Description
Aconitase Assay Kit (BA0070) (BA0070)
The Aconitase Assay Kit (SKU: BA0070) provides a fast, sensitive and high-throughput method for measuring aconitase activity in biological samples. Aconitase (aconitate hydratase) is an enzyme in the citric acid (TCA) cycle that catalyses the conversion of citrate to isocitrate, and its activity depends largely on the iron-sulfur cluster. Related conditions include aconitase deficiency, Friedreich's ataxia and diabetes. In this assay the isocitrate generated by the aconitase reaction is oxidised to produce NADPH, which converts a dye to an intense violet colour with an absorption maximum at 565 nm. The increase in absorbance at 565 nm is directly proportional to aconitase activity, and the homogeneous format can be readily automated for thousands of samples per day.
| Product Name: | Aconitase Assay Kit (BA0070) |
| SKU: | BA0070 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.5 to 100 U/L for a 20 minute reaction |
| Sample Type: | Cell lysate, tissue homogenate, serum and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 30 minutes (readings at 10 and 30 minutes) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric determination of aconitase activity. Isocitrate generated by aconitase is oxidised to produce NADPH, which converts a dye to a violet colour measured at 565 nm.
- Fast and sensitive
- Linear detection range 0.5 to 100 U/L for a 20 minute reaction using 20 uL sample
- Convenient homogeneous mix-incubate-measure format
- Robust and amenable to high-throughput screening
- Aconitase activity determination in 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 phosphate buffered saline (pH 7.4), homogenise 50 mg tissue in about 200 uL cold PBS and centrifuge at 800 x g for 10 minutes at 4C. For cells, collect by centrifugation, homogenise or sonicate in cold PBS and centrifuge at 800 x g. For the mitochondrial fraction, centrifuge the supernatant at 20,000 x g for 10 minutes at 4C, resuspend the pellet in cold PBS and sonicate for 20 seconds. |
| 2 | Reagent preparation. Keep thawed Enzyme A and B on ice and equilibrate other reagents to 25C. |
| 3 | Standards. Prepare 200 uL 5000 uM premix by mixing 10 uL Standard (100 mM) with 190 uL distilled water and prepare the dilution series shown in the table. Transfer 20 uL standards into wells of a clear flat-bottom 96-well plate. Transfer 20 uL of each sample into separate wells; samples with high dehydrogenase activity or isocitrate need a Sample Blank. |
| 4 | Working reagent. For each well mix 8 uL NADP/MTT, 1 uL Enzyme A, 1 uL Enzyme B, 5 uL Substrate and 70 uL Assay Buffer (for Sample Blanks omit Enzyme A and Substrate, using 8 uL NADP/MTT, 1 uL Enzyme B and 75 uL Assay Buffer). Add 80 uL working reagent to each well and tap to mix. |
| 5 | Incubate at room temperature and read OD at 565 nm at 10 minutes and 30 minutes. |
Subtract the blank value (water, standard #4) from the standard values at 10 minutes and plot the change in OD against standard concentrations to determine the slope. Aconitase activity = (OD30 - OD10) / (t x slope) x n U/L, where t is the reaction time (20 minutes) and n is the dilution factor. One unit catalyses the conversion of 1 umole of citrate to isocitrate per minute at pH 7.4. If activity exceeds 100 U/L, dilute the sample, repeat and multiply by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| NADP/MTT | 1 mL | -20C |
| Standard | 1 mL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| Substrate | 1 mL | -20C |
