Overview

  • Product name

    Isocitrate Assay Kit
  • Detection method

    Colorimetric
  • Sample type

    Cell Lysate, Tissue Lysate, Food samples
  • Assay type

    Quantitative
  • Sensitivity

    > 0.2 µg
  • Range

    0.2 µg - 5 µg
  • Assay time

    0h 40m
  • Product overview

    Isocitrate Assay Kit (ab83424) provides a simple, sensitive and rapid means of quantifying isocitrate in a variety of samples. In this assay, isocitrate is oxidized with the generation of NADPH which converts a nearly colorless probe to an intensely colored species with a λmax of 450nm. This assay can detect 1 to 20 nmoles (~0.2-5 µg) of isocitrate.
    Visit our FAQs page for tips and troubleshooting.

  • Notes

    Isocitric acid (HOOC-CHOH-CH (-COOH)-CH2-COOH) is an intermediate of the Krebs TCA cycle, positioned between citrate and α-keto-glutarate. It is the branch point from which the glyoxylate shunt operates in plants and lower organisms. Isocitrate is found in substantial concentrations in many fruits and vegetables as well as in foods produced from these raw materials. In the TCA cycle, isocitrate is oxidized by isocitrate dehydrogenase (IDH) to α-ketoglutarate with the generation of NAD(P)H. Loss of NAD-IDH has been implicated as a potential causative factor in retinitis pigmentosa.

  • Platform

    Microplate reader

Properties

Associated products

Images

  • Isocitrate standard curve generated using this kit protocol.

Protocols

References

ab83424 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer

Thank you for your enquiry. I would be pleased to help answer your questions. 1. I can confirm you should be able to use these kits with blood samples. Generally serum or plasma that is obtained from blood samples is used for such purposes. However if you wish to use whole blood you will need to lyse the red blood cells in in each kits assay buffer and some optimization will be required by the customer in this case. 2. Overall you can use the following general procedure to prepare the sample. Samples can be deproteinized if indicated in respective data sheets either by using the PCA method or by using 10 kDa spin cut-off filters. For cell samples: i. Start with ~2X106 cells, suspend the cell pellet 500 μl (or ~4 volumes) of the assay buffer on ice. ii. Homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. iii. Spin down the sample and collect the supernatant. iv. Load the supernatant unto a 10 kda spin column for deproteinzation (please use this step only if indicated in the protocol provided with the online data sheet- this step is NOT required for enzyme assays). v. Use the eluate for the subsequent assays. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve. 3. I am sorry to conrirm we do not sell the cut of columns or deprotinization kits. I would like to recommend checking the Biocompare website which has an excellent search facility that includes many suppliers. The links are: www.biocompare.com http://www.biocompare.com/ProductCategories/2045/Antibodies.html I hope this will be helpful to you. Should you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for your enquiry regarding ab83396 and ab83424. Yes, you can use these kits with blood samples. I would suggest obtaining serum / plasma from the blood sample before proceeding with the assay. For blood plasma- “Blood plasma is prepared by spinning a tube of fresh blood containing an anti-coagulant in a centrifuge until the blood cells fall to the bottom of the tube. The blood plasma is then poured or drawn off.” For serum samples- “For separating serum from the blood cells you should use a tube without any anticoagulant , sterile empty tube will be fine , after taking your sample , say 5 ml or so leave the tube in a standing position for about 20-30 minutes ( it can take shorter time than this so you can check it periodically ) after that you will find your blood to be clotted , centrifuge at 20 c degree , 1500g for 10 minutes then remove your serum very quickly and flash freeze it in -80 0C.” I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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