Aconitase Enzyme Activity Microplate Assay Kit (ab109712)

Overview

• Product nameAconitase Enzyme Activity Microplate Assay KitSee all Aconitase kits ...
• Tests
  1 x 96 well plate
• Sample type
  Cell culture extracts, Tissue
• Assay typeEnzyme activity
• Assay time
  2h 00m
• 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.

• Notes

  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.

  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

Properties

• 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
  • Metabolism
  • Pathways and Processes
  • Metabolic signaling pathways
  • Energy transfer pathways
  • Energy Metabolism

  • Kits/ Lysates/ Other
  • Kits
  • Cell Damage Kits
  • Oxidative stress

  • Kits/ Lysates/ Other
  • Kits
  • Cell Metabolism Kits
  • Other Metabolism Assay

  • Signal Transduction
  • Metabolism
  • Energy Metabolism

Applications

Aconitase Enzyme Activity Microplate Assay Kit images

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  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.
• 

  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.

Protocols

Protocol Booklet