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Product name
Aconitase Enzyme Activity Microplate Assay Kit
See all Aconitase products (2) ...
Tests
1 x 96 well plate
Sample type
Cell culture extracts, Tissue
Assay type
Enzyme activity
Assay time
2h 00m
Product overview
ab109712 Aconitase Enzyme Activity Microplate Assay Kit measures the activity of aconitase in a sample by following the conversion of isocitrate to cis-aconitate as an increase in absorbance at UV 240 nm with an extinction coefficient of 2.2 OD/mM per well. Aconitase preservation solution, assay buffer, reagents and a special UV plate are provided for this measurement by a UV 240nm capable spectrophotometer.
Aconitase is an iron-sulfur protein that catalyzes the reversible interconversion 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 mitochondrial (ACO2) and cytosolic (ACO1) 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. Both forms are sensitive to oxidants, which can inactivate the enzyme by changing the [4Fe-4S] to a [3Fe-4S] cluster. This change in mitchondrial aconitase can lead to 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.
An inactivated [3Fe-4FS] aconitase may be activated in vitro by the addition of iron and cysteine. The active form of the enzyme is inhibited by citrate analogs, fluoracetate, and redox stress agents. Other inhibitors include oxidative stress agents such as peroxynitrite, hydrogen peroxide and superoxide, which lead to [3Fe-4FS] inactivation of the enzyme. Therefore, aconitase is considered a good marker of mitochondrial and cellular oxidative stress.
Tested applications
Functional Studiesmore details
Properties
Storage instructions
Store UV microplate at room temperature. Store all other components at 4°C.
| Components | 96 tests |
|---|---|
| 96-well UV microplate | 1 unit |
| Aconitase preservation solution | 1 x 20ml |
| Buffer | 1 x 50ml |
| Detergent (for cultured cell preparation only) | 1 x 1ml |
| Isocitrate (25X) | 1 x 800µl |
| Manganese (100X) | 1 x 200µl |
Research areas
Metabolism >> Pathways and Processes >> Metabolic signaling pathways >> Energy transfer pathways >> Energy Metabolism
Kits/ Lysates/ Other >> Kits >> Cell Damage Kits >> Oxidative stress kits
Kits/ Lysates/ Other >> Kits >> Cell Metabolism Kits >> Other Metabolism Assay
Signal Transduction >> Metabolism >> Energy Metabolism
Relevance
Aconitase (aconitate hydratase; EC 4.2.1.3) 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 localization
ACO1: Cytoplasmic ACO2: Mitochondrial
Alternative names
- ACO 1
- ACO 2
- ACO1
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Applications
Show applications key
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.
Functional Studies
FuncSFuncS
Aconitase Enzyme Activity Microplate Assay Kit images:
ELISA - Aconitase Enzyme Activity Microplate Assay Kit (ab109712)
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.
ELISA - Aconitase Enzyme Activity Microplate Assay Kit (ab109712)
Figure 2. Using cellular fractionation kit ab109719, whole cells were separated into cytoplasmic, mitochondrial and nuclear fractions.
Protocols
References for Aconitase Enzyme Activity Microplate Assay Kit (ab109712)
This product has been referenced in:
- 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) »
- Celotto AM et al. Modes of metabolic compensation during mitochondrial disease using the Drosophila model of ATP6 dysfunction. PLoS One 6:e25823 (2011).Read more (PubMed: 21991365) »
See all 2 publications for this product
Publishing research using ab109712? Please let us know so that we can cite the reference in this datasheet
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"