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Product Details
| Cat # +Size | K716-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance at 450 nm |
| Species Reactivity | Mammalian |
| Applications | N/A |
| Features & Benefits | • Simple procedure; takes ~ less than 40 minutes • Fast and convenient |
| Kit Components | • Assay Buffer • Substrate • Developer • Enzyme Mix • Cysteine-HCl • (NH₄)₂Fe(SO₄)₂ • Isocitrate Standard |
| Storage Conditions | 4°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Aconitase (aconitate hydratase; EC 4.2.1.3) is an iron-sulfur protein containing [Fe₄S₄]²ᶧ cluster that catalyzes the stereospecific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process. Tissue contains two aconitases, a mitochondrial (m-) and a cytosolic (c-) aconitase. They are related, but distinctly different enzymes and are coded for on different chromosomes. Loss of aconitase activity in cells or other biological samples treated with pro-oxidants has been interpreted as a measure of oxidative damage. BioVision’s Aconitase Assay Kit is a highly sensitive, simple, direct and HTS-ready colorimetric assay for measuring Aconitase activity in biological samples. In the assay, citrate is converted by aconitase into isocitrate, which is further processed resulting in a product that converts a nearly colorless probe into an intensely colored form with a λmax at 450nm.
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Why do you need the activation mixture?
The reason for needing to activate the aconitase in the sample is as follows: The Iron sulphur cluster required for Aconitase activity is inactivated in biological samples when the aconitase is exposed to oxidants/superoxides. The way to measure Aconitase activity in vitro requires reinsertion of ferrous iron into the iron sulphur cluster. This is why you need to use the activation solution, so that the Aconitase in the samples can be tested for activity. This is a well-studied and documented process reported in literature: http://www.jbc.org/content/258/18/11098.full.pdf.
Sample preparation procedure: there are 2 centrifuge steps one at 800 g and then at 20,000 g. Customer do not have a 20000 g centrifuge for centrifuging small volume. What is the alternative? Can they increase the time from 15 min to ? for getting a pellet yield similar to centrifuging at 20000g for 15 mins. Any suggestion is helpful.
The g-force at this step is important since the 20,000g spin fractionates mitochondria at this step. The customer needs to find a centrifuge that can spin at 20,000 g to be able to pellet mitochondria. Otherwise the m-aconitase activity cannot be specifically estimated. Increasing time at a lower speed will not yield mitochondria effectively.
Can the kit measure the activity of mitochondrial (m-) and a cytosolic (c-) aconitase separately?
While measuring aconitase acitvities in cell lysates, the kit cannot distinguish between the different fractions. Therefore, to measure cytosolic and mitochondrial aconitase acitivities separately, they need to collect the two fractions and then perform the assay in each fractions.
I added protease inhibitor to assay buffer for sample preparation. Is there any possibilites that the protease inhibitor affected on measurement of aconitase?
Aconitase is an isomerase and hence the protease inhibitors should not affect the assay.
Can I omit the sonication step?
The sonication step is crucial to make sure that the mitochondrial pellet is homogeneously mixed in the buffer. If you set the microtip sonicator at 50-60% intensity for 20 secs, it would automatically stop at 20 secs after emanating the exact same # of pulses. Alternately manual homogenization can be used but this is more time consuming and inconsistent between samples.
The last wash step in my mito isolation protocol uses EDTA. Will this be a problem for the Assay ?
Aconitase enzyme has an iron-sulphur cluster this is critical for its activity. Washing the purified mitochondrial fractions with EDTA (a metal chelator) is not a good idea. The assay buffer contains some detergent (<0.5% NP-40) to help lyse cells.
Can we use frozen samples with this assay?
We would recommend using fresh samples. However, frozen samples can be used too provided they have not undergone multiple freeze thaw cycles.
Can the kit work on bacteria or yeast cells?
The kit has been standardized for mammalian cells only.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
BioVisions’s assay kits expire 6-12 months from the date of shipment. Some components of the kit may expire sooner as mentioned in the data sheet.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Does the Assay Buffer lyse the cells? If it does, does it only disrupt the cell membrane, or are mitochondrial / nuclear membranes also affected?
The detergents used in the assay buffer are a non-ionic and non-denaturing in nature. Thus, it will only lyse the cell membrane and not nuclear or mitochondrial membranes.
Can you please provide the detection limit for the Aconitase Assay Kit, K716-100?
The lower limit of detection is calculated by taking 2 nmol (lowest amount of Standard) divided by 50 µl (max sample volume) = 2 nmol/50µl = 0.04 nmol/µl = 40 µM
The highest limit of detection is calculated by taking 20 nmol (highest amount of Standard) divided by 2 µl (min sample volume) = 20 nmol/2 µl = 10 nmol/µl = 10,000 µM
The highest limit of detection is calculated by taking 20 nmol (highest amount of Standard) divided by 2 µl (min sample volume) = 20 nmol/2 µl = 10 nmol/µl = 10,000 µM
Wu, Lei et al. (2017) Lack of β, β-carotene-9′, 10′-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice, Mol Nutr Food Res. 2017 May;61(5).
Pruszynska-Oszmalek et al., In ovo injection of prebiotics and synbiotics affects the digestive potency of the pancreas in growing chickens. Poultry Science, Aug 2015; 94: 1909 - 1916.
Ping La et al., Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation Stabilizes ISCU Protein: IMPLICATIONS FOR IRON METABOLISM. J. Biol. Chem., May 2013; 288: 12901 - 12909.
For more citations of this product click here
