Aconitase Enzyme Activity Microplate Assay Kit (ab109712)
- Product nameAconitase Enzyme Activity Microplate Assay KitSee all Aconitase kits ...
- Detection methodColorimetric
- Tests1 x 96 well plate
- Sample typeCell culture extracts, Tissue
- Assay typeEnzyme activity
- Assay time2h 00m
- Assay durationMultiple steps standard assay
- Product overview
ab109712 is a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates. Unlike other assays this is not a coupled reaction and therefore only aconitase activity is required and measured. Aconitase catalyzes an equilibrium between aconitate, cis-aconitate and iso-citrate. These reactions are monitored by measuring the increase in absorbance at 240 nm associated with the formation of the cis-aconitate which has an extinction coefficient of 2.2 OD/mM per well. Therefore the rate of cis-aconitate production is proportional to aconitase activity.
Aconitase preservation solution, assay buffer, reagents and an essential UV microplate are provided for this measurement. The entire assay can be completed within 2 hours.
Note – mitochondrial and cytoplasmic aconitase activities are indistinguishable. Therefore, to measure the mitochondrial activity only, first isolate mitochondria, or for both activities fractionate the cells into cytoplasmic and mitochondrial.
Aconitase (aconitate hydratase; EC 126.96.36.199) is an iron-sulfur protein that catalyzes the reversible inter-conversion of citrate and isocitrate, via a cis-aconitate intermediate, in both the TCA and glyoxylate cycles. The enzyme contains a [4Fe-4S] cluster which interacts directly with the substrates. In eukaryotes there are both mitochondrial and cytosolic forms of the enzyme. The mitochondrial form functions not only in the TCA cycle, but also to stabilize mtDNA thereby influencing mitochondrial gene expression. The cytosolic form can function as an aconitase as well as an iron regulatory protein.
The active form of the enzyme is inhibited by citrate analogs, and fluoracetate. Other inhibitors include oxidative stress agents such as peroxynitrite, hydrogen peroxide and superoxide, which inactivate the enzyme by changing the [4Fe-4S] to a [3Fe-4S] cluster. Aconitase is considered a good marker of mitochondrial and cellular oxidative stress. This change in mitochondrial aconitase can lead to a decreased energy production, whereas in cytosolic aconitase it triggers binding of the enzyme to mRNA iron response elements resulting in increased expression of iron uptake proteins and decreased transcription of iron sequestering protein.
A hydroxyl scavenging solution (Aconitase preservation solution) is supplied with this kit to maintain aconitase activity during sample preparation. An inactivated [3Fe-4FS] aconitase may be activated in vitro by the addition of iron and cysteine.
- Tested applicationsFunctional Studies more details
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 96 tests 10X Detergent 1 x 1ml 96-well UV microplate 1 unit Aconitase preservation solution 1 x 20ml Buffer 1 x 50ml Isocitrate (25X) 1 x 800µl Manganese (100X) 1 x 200µl
- Research Areas
- RelevanceAconitase (aconitate hydratase; EC 188.8.131.52) is an iron-sulfur protein containing an [Fe4S4]2+ 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 prooxidants has been interpreted as a measure of oxidative damage.
- Cellular localizationACO1: Cytoplasmic ACO2: Mitochondrial
- ACO 1
- ACO 2
- Aconitase 1 soluble
- Aconitase 2 mitochondrial
- Aconitate hydratase
- Aconitate hydratase mitochondrial
- Citrate hydro lyase
- Cytoplasmic aconitate hydratase
- EC 184.108.40.206
- Ferritin repressor protein
- IRE BP 1
- Iron regulatory protein 1
- Iron responsive element binding protein 1
Our Abpromise guarantee covers the use of ab109712 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Aconitase Enzyme Activity Microplate Assay Kit images
Figure 2. Using cellular fractionation kit ab109719, whole cells were separated into cytoplasmic, mitochondrial and nuclear fractions.
Figure 1. Bovine heart mitochondria were treated with increasing concentrations of hydrogen peroxide and peroxynitrite to generate aconitase activity IC50's for these oxidative stress agents.
References for Aconitase Enzyme Activity Microplate Assay Kit (ab109712)
This product has been referenced in:
- Jiang H et al. Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration. Int J Radiat Oncol Biol Phys N/A:N/A (2012). Read more (PubMed: 22975619) »
- Choi EM & Lee YS Involvement of PI3K/Akt/CREB and redox changes in mitochondrial defect of osteoblastic MC3T3-E1 cells. Toxicol In Vitro 25:1085-8 (2011). Read more (PubMed: 21466842) »