Aconitase Assay Kit

(Catalog #K716-100; 100 reactions; Store kit at –20^oC)

1. 
   

   Introduction:

   
   

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 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 λ_max at 450nm.

2. 
   

   Kit Contents:

   
   

Components	K716-100	Cap Code	Part No.
Assay Buffer Substrate Developer Enzyme Mix (200µl) Cysteine (NH4)2Fe(SO4)2 Isocitrate Standard (10µmol)	30 ml lyophilized lyophilized 200 µl lyophilized lyophilized lyophilized	WM Blue Purple Green Red Brown Yellow	K716-100-1 K716-100-2 K716-100-3 K716-100-4 K716-100-5 K716-100-6 K716-100-7

3. 
   

   Storage and Handling:

   
   

Store the kit at -20^oC, protect from light. Warm the assay buffer to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay.

4. 
   

   Reagent Reconstitution and General Consideration:

   
   

Substrate: Dissolve with 220µl DiH₂O; sufficient for 100 assays.

Developer: Dissolve with 1.1ml Assay Buffer before use; sufficient for 100 assays.

Aconitase Activation Solution: Dissolve cysteine and (NH₄)₂Fe(SO₄)₂ with 0.5ml Assay Buffer separately, and store at -20°C. Take out 0.1ml cysteine and (NH₄)₂Fe(SO₄)₂ solutions and mix together to prepare fresh activation solution.

All the solutions except the activated Aconitase are stable for 1 month at -20°C.

Ensure that the Assay Buffer is at room temperature before use. Keep samples, Enzyme Mix and Aconitase solution on ice during the assay.

5. 
   

   Aconitase Activity Assay:

   
   

0. 
   

   Sample Preparations:
   
   Homogenize 20-40 mg tissue or 10⁶ Cells on ice in 0.1 ml cold Assay Buffer; Centrifuge at 800 x g for 10 min at 4°C; Collect the supernatant for c-aconitase assay. For m-aconitase assay, centrifuge the supernatant at 20,000 x g for 15 min at 4°C and collect the pellet, dissolve into 0.1 ml cold Assay Buffer, sonicate for 20 seconds. Keep samples at -80°C for storage.

   
   

Add 10 µl activation solutions to 100 µl sample; incubate on ice for 1 hour to activate aconitase in the sample.

Add 2-50µl activated samples into each well, and adjust volume to 50µl. We suggest using a background control group as well as several doses of your sample to ensure the readings are within the linear range.

1. 
   

   Isocitrate Standard Curve:
   
   Dissolve into 0.5ml assay buffer to prepare 20mM isocitrate standard solution. Take 20µl 20mM standard solution and add 180µl assay buffer to prepare 2mM isocitrate standard solution. Add 0, 2, 4, 6, 8, 10µl 2mM Isocitrate Standard solution into 96-well plate in duplicate to generate 0, 4, 8, 12, 16, 20 nmol/well Isocitrate standard. Bring the final volume to 50µl with Assay Buffer.

   
   


2. 
   

   Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix:

   
   

Sample Reaction Mix Background Mix

46 μl Assay Buffer 48 μl Assay Buffer

2 μl Enzyme Mix 2 μl Enzyme Mix

2 μl Substrate

Add 50 μl of the Sample Reaction Mix to each test samples, background control and Isocitrate standards. Mix well and incubate at 25°C for 30-60 min. Add 10μl Developer to each well, mix and incubate at 25°C for 10min. Measure at O.D.450nm.

3. 
   

   Calculation: Plot the Isocitrate standard curve. ∆OD=OD_sample-OD_background, apply the ∆OD to the Isocitrate standard curve to get B nmol of isocitrate generated by aconitase in 30-60min.

   
   

Aconitase Activity = Sample Dilution Factor = nmol/min/ml = mU/mL

Where: B is the isocitrate amount from standard Curve (in nmol).

T is the time incubated.

V is the pretreated sample volume added into the reaction well (in ml).

One unit will isomerize 1.0 μmol of Citrate to Isocitrate per min at pH 7.4 at 25 °C.