Overview

  • Product name

    Citrate Synthase Activity Assay Kit
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    1 3 5.45%
    Inter-assay
    Sample n Mean SD CV%
    1 3 8.3%
  • Sample type

    Cell culture extracts, Tissue Extracts
  • Assay type

    Enzyme activity
  • Sensitivity

    1.56 µg/ml
  • Range

    1.56 µg/ml - 100 µg/ml
  • Assay time

    3h 15m
  • Species reactivity

    Reacts with: Human
    Does not react with: Mouse, Rat
  • Product overview

    Citrate Synthase Activity Assay Kit (ab119692) is used to determine mitochondrial citrate synthase (CS) activity in a sample. The enzyme is immunocaptured within the wells of a microplate, and activity is determined by recording color development of TNB, which is generated in the reaction of citrate synthesis. The reaction product, TNB, absorbs at OD = 412 nm.


    This kit immunocaptures only native citrate synthase from the samples. Using the immunocapture method allows the measurement of citrate synthase activity with simple sample preparation and no need for mitochondrial isolation.


    This kit can be used with whole cell extracts or tissue homogenates.


    Citrate synthase assay protocol summary:
    - add samples to wells
    - incubate for 3 hrs to capture enzyme
    - wash with wash buffer
    - add activity solution
    - analyze with microplate reader for 5-30 min at 20s intervals

  • Notes

    Citrate synthase (CS, O75390) is the initial enzyme of the tricarboxylic acid (TCA) cycle. This enzyme is a 51.7 kDa enzyme that catalyzes the reaction of 2 carbon acetyl CoA with 4 carbon oxaloacetate to form the 6 carbon citrate (EC 2.3.3.1). This enzyme is an exclusive marker of the mitochondrial matrix. Citrate synthase is found in nearly all cells capable of oxidative metabolism.

    Storage: All components are shipped cold. 10X Reaction Reagent mixture is shipped lyophilized. Before use, rehydrate the reaction mixture by adding 1mL base buffer. Vortex the tube thoroughly to dissolve the lyophilized powder to prepare 10X Reaction solution. After hydration unused amounts of the 10X Reaction solution should be stored at -80°C for 6 months. Store all other components at 4°C. This kit is stable for 6 months from receipt.

  • Platform

    Microplate reader

Properties

Images

  • Figure 2. Example activity measurements from titrated of Human heart homogenate.
  • Figure 1. Example activity measurements from titrated HepG2 cells.

Protocols

References

This product has been referenced in:

  • Zhao Z & Pu Y Lixisenatide enhances mitochondrial biogenesis and function through regulating the CREB/PGC-1a pathway. Biochem Biophys Res Commun 508:1120-1125 (2019). Read more (PubMed: 30553453) »
  • Corbet C  et al. Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects. Nat Commun 9:1208 (2018). Read more (PubMed: 29572438) »
See all 11 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Abreviews
This kit was purchased to see if it was compatible with mitochondria isolated from bovine PBMCs. The initial data looks good but more tests are needed to see if bovine samples can be stored at -80C for 6 months like stated in the protocol booklet. The image I attached shows the following data: A1 B1 (Blank), C1 D1 (Fresh Sample A), E1 F1 (Fresh Sample B), G1 H1 (1 month old sample C). There was some variation in the methods used to isolate the mitochondria in the fresh and 1 month old sample as we are still working to improve this part of the experiment. It is unknown if the variation in mitochondria isolation methods or the age of the samples used is responsible for the differences observed graphically.

Abcam user community

Verified customer

Submitted Sep 27 2016

Answer

1. This kit passed QC test so all the components should be prepared properly.
2. Try using a higher protein concentration (e.g. 500ug/ml in lieu of 100ug/ml), since different cell lines have different expression level of CS.
3. Increase the incubation time of the incubation buffer to O/N rather than 3hr.

This kit works only with human samples. I could not find a proper positive control in our catalog, we tested this kit with the following materials which can be used as positive controls. These are HepG2 cells, HDFn cells (other fibroblast cells should also work), human liver homogenate, and human heart homogenate.

I hope this information is helpful to you and yields improved results. Please do not hesitate to contact us with any further questions.

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Answer

Thank you for contacting us.

Same process for converting all to molarity per amount of protein loaded into well.
Note these are all per cm and should be converted into path length for microplate which should be about 0.6cm.

Extinction coefficients

ab109908 (complex II enzyme activity),
ε600 = 21 mM-1cm-1

ab109909 (complex IV activity),
ε550 = 21.1 mM-1cm-1

ab109716 (ATP synthase activity)
ε340 = 6.220 mM-1cm-1

ab119692 (citrate synthase activity).
Ε412 = 14.1 mM-1cm-1

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

I have received word back from the lab regarding your request. Unfortunately the details of the reagents and reactions for these products are proprietary and we cannot share them. They have provided what details of the citrate synthase and PDH system which they are able to. I will attach these to this email.

These products are covered by our Abpromise guarantee. We provide scientific support, replacement or refund should this product not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Answer

Thank you for contacting us.

The composition of the extraction buffer (P/N 8201093) is Hepes, NaCl, and a detergent, pH 7.4. Thus, it does not contain any of the other components you are concerned about (Tris-HCl, KPO4, or EDTA).

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us. You may be interested in the following kits:

Citrate Synthase: ab119692
Aconitase: ab109712 or ab83459
CitrateLyase: wedo not currently have a kit available, but weyou may be interested in ourprimary antibodies ab40793 and ab61762
NAD/NADH: ab65348 (we also havea kit forNADH Dehydrogenase: ab124535)
Isocitrate Dehydrogenase: ab102528
Isocitrate Lyase: no kits or antibodies currently available

I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for contacting us.

Yes you can this kit uses lauryl maltoside to extract active enzymes.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

I have heard back from the lab with the following information:

All these assays require different sample concentrations described in each booklet protocol - but in most cases is approximately 10ug/microplate well.
The good news is that normal whole muscle homogenate contains a good robust mitochondria signal so less is required, muscle samples are described in most if not all protocols.
Another good news is that the same preparation for any of the assays is suitable for all others. I would estimate that if a 1 mg of homogenate can be prepared (0.2mL at 5mg/mL).
This would provide enough material for each of the assays when diluted to the specific concentration in each assay.
We would recommend - if contemplating doing multiple assays - to include a mitochondrial sample as positive control such as ab110338 isolated mitochondria (note this is cow source)



I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for your inquiry.


Unfortunately, this kit does not work in mouse. The capture antibody works in human but not in rat or mouse. Other species have not been tested yet.


This is the only Citrate Synthase Activity Assay Kit we carry. You may want to try http://www.biocompare.com to see if there is another commercially available.


I am sorry that this information could not be more helpful. Please contact us with any other questions.

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