Caspase-9 Inhibitor Assay Kit (ab102497)

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Overview

  • Product name

    Caspase-9 Inhibitor Assay Kit
    See all Caspase-9 kits

  • Detection method

    Fluorescent

  • Sample type

    Adherent cells, Suspension cells

  • Assay type

    Quantitative

  • Assay time

    1h 30m

  • Product overview

    Abcam's Caspase 9 Inhibitor Drug Detection Kit provides an effective means for screening caspase inhibitors using fluorometric methods. The assay utilizes synthetic peptide substrate LEHD-AFC (AFC, 7-amino-4-trifluoromethyl coumarin). Active caspase 9 cleaves the synthetic substrate to release free AFC which can then be quantified by fluorometry. Compounds to be screened can directly be added to the reaction and the level of inhibition of caspase 9 activity can be determined by comparison of the fluorescence intensity in samples with and without the testing inhibitors.
    Visit our FAQs page for tips and troubleshooting.

  • Notes

    Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. Inhibition of caspases can delay apoptosis, implicating a potential role in drug screening efforts.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    2X Reaction Buffer 1 x 10ml
    Active Caspase-9 1 x 100 units
    Caspase Inhibitor, Z-VAD-FMK (2 mM) 1 x 10µl
    Caspase Substrate LEHD-AFC (1 mM) 1 x 0.5ml
    DTT (1 M) 1 x 100µl

  • Research areas

  • Relevance

    Caspases are cysteine proteases, expressed as inactive precursors, that mediate apoptosis by proteolysis of specific substrates. Caspases have the ability to cleave after aspartic acid residues. There are two classes of caspases involved in apoptosis; initiators (activation by receptor cluster) and effectors (activation by mitochondrial permeability transition). Proapoptotic signals autocatalytically activate initiator caspases, such as Caspase 8 and Caspase 9. Activated initiator caspases then process effector caspases, such as Caspase 3 and Caspase 7, which in turn cause cell collapse. Caspase 9 (also known as ICE like apoptotic protease 6 (ICE LAP6), apoptotic protease Mch6, and apoptotic protease activating factor 3 (Apaf3)) is a member of the peptidase family C14 that contains a CARD domain. It is active as a heterotetramer and has been reported to have two isoforms. ProCaspase 9 is approximately 47 kD. It is present in the cytosol and, upon activation, translocates to the mitochondria. Caspase 9 is involved in the caspase activation cascade responsible for apoptosis execution and cleaves/activates Caspase 3 and Caspase 6. It becomes activated when recruited to the Apaf1/cytochrome c complex.

  • Alternative names

    • APAF3
    • CASP9
    • Caspase 9
    • ICE LAP6
    • MCH6
    see all

Images

  • Functional assays: Caspase 9 Inhibitor Drug Detection Kit (ab102497)
    Functional assays: Caspase 9 Inhibitor Drug Detection Kit (ab102497)

    Titration of the caspase 9 inhibitor Z-LEHD-FMK (ab142026) (duplicates; +/- SD).

  • Functional assays: Caspase 9 Inhibitor Drug Detection Kit (ab102497)
    Functional assays: Caspase 9 Inhibitor Drug Detection Kit (ab102497)

    Titration of the caspase inhibitors Z-VAD(OMe)-FMK (ab120487) and Q-LEHD-Oph (ab142039) (duplicates; +/- SD).

  • Functional assays: Caspase 9 Inhibitor Drug Detection Kit (ab102497)
    Functional assays: Caspase 9 Inhibitor Drug Detection Kit (ab102497)

    Titration of caspase inhibitor Z-VAD-FMK (ab102497) (duplicates; +/- SD).

  • Caspase 9 Inhibitor Drug Detection Kit
    Caspase 9 Inhibitor Drug Detection Kit

    Caspase activity (RFU) in presence of 0µM - 40µM of z-VAD-FMK (generic caspase inhibitor), assessed using LEHD-AFC as caspase 9 substrate and following Caspase 9 Inhibitor Drug Detection Kit (ab102493) protocol. Lanes:

    1.- Background Control: no Caspase 9, no z-VMAD-FMK

    2.- Positive Inhibition Control: no Caspase 9, +z-VAD-FMK

    3.- Positive Control: + Caspase 9, no z-VAD-FMK

    4.- Caspase 9 + 2.5µM z-VAD-FMK

    5.- Caspase 9 + 10µM z-VAD-FMK

    6.- Caspase 9 + 40µM z-VAD-FMK

References (0)

Publishing research using ab102497? Please let us know so that we can cite the reference in this datasheet.

ab102497 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

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Question

Hi,

I had another question about the Caspase 9 Inhibitor Drug Detection Kit (ab102497). I have run the assay and the RFUs for the positive inhibition are about 10x the RFUs for the background control. Is this expected? I have not tried higher concentrations of VAD-FMK; should the concentration of VAD-FMK specified in the protocol fully inhibit caspase 9 activity? Which control would you suggest using for normalization?

Thank you,

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Answer

Thank you very much for your email and for your patience while I've been in touch with the lab regarding your enquiry.

My contact at the lab has informed me that the background control is the fluorescence emitted by the substrate and the reaction buffer. These values have to be subtracted from all values. Your signals from the active caspase activity should be taken as 100% activity and used for normalization. The signals you then get from the use of our caspase inhibitor will give you an idea of how inhibition looks and what kind of signals it produces. This can be reported in terms of % inhibition. Then you can use your experimental inhibitor to looks at the % inhibition it produces.
I hope that this information will be useful, but if you have further questions or need anything else, please let me know and I'll be happy to help.

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Question

The volumes in the protocol for ab102497 looks to be for a 96-well plate. Are there any reported problems with decreasing volumes for use in a 384-well plate (if this has been explored)? Also from the website it is not clear what the concentration of Z-VAD-fmk is used. What is the stock concentration of Z-VAD-fmk? Just to clarify the 1uL Z-VAD-fmk into 49uL H20 is used for a completely inhibited control?

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Answer

Thank you for your inquiry.

1) We have not looked at decreasing volumes for 384-well plates. You may be
able to use these, but it will depend on the amount of caspase in the samples
and hence signal strength can be impacted.

2) The Caspase Inhibitor, Z-VAD-FMK is 2 mM. I have since updated the online datasheet.

Hence 1 ul in 50 ul would result in 40 uM; which should be adequate for
recombinant Caspases. If you're looking at cell lystaes, you should probably
increase Z-VAD-FMK final to about 2-5x.
The updated component list will look like this, but it takes a few days to update:


Components 1 kit
2X Reaction Buffer 1 x 10ml
Active Caspase-9 1 x 100 units
Caspase Inhibitor, Z-VAD-FMK (2 mM) 1 x 10µl
Caspase Substrate LEHD-AFC (1 mM) 1 x 0.5ml
DTT (1 M) 1 x 100µl



I hope this information helps. Please contact us with any other questions.

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