Aconitase Assay Kit (Colorimetric)

Product details

Aconitase Assay Kit ab109712 is a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates.

Aconitase assay principle
In the aconitase assay protocol, aconitase catalyzes an equilibrium between citrate, 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. The rate of cis-aconitate production is proportional to aconitase activity.

The active form of aconitase 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.
A hydroxyl scavenging solution (Aconitase preservation solution) is supplied with this aconitase assay kit to maintain aconitase activity during sample preparation.
An inactivated [3Fe-4FS] aconitase may also be activated in vitro by the addition of iron and cysteine (not provided in this kit).

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 assay protocol summary
- prepare samples and used aconitase preservation solution to stabilize aconitase activity
- add samples and reagents to plate
- incubate at room temperature
- readout on a colorimetric plate reader at 0-min and 30-min

Related Aconitase assay kits< br/> For an alternative aconitase assay method, based on an enzyme-coupled reaction with isocitrate dehydrogenase, use Aconitase assay kit ab83459. ab83459 is our most popular Aconitase assay kit.

Other notes

Previously called Aconitase Enzyme Activity Microplate Assay Kit and Aconitase Activity Assay Kit.

Aconitase (aconitate hydratase; EC 4.2.1.3) 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.