Cytochrome c Release Assay Kit (ab65311)

Reviews (3)Q&A (9)References (17)

Overview

  • Product name

    Cytochrome c Release Assay Kit
    See all Cytochrome c kits

  • Sample type

    Tissue, Adherent cells, Suspension cells

  • Assay type

    Direct

  • Assay time

    3h 00m

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Product overview

    Cytochrome c Release Assay Kit ab65311 provides an effective means for detecting cytochrome c translocation from mitochondria into cytosol during apoptosis.


    The kit provides reagents to isolate a highly enriched mitochondria fraction from cytosol. The procedure is simple and easy to perform; no ultracentrifugation is required and no toxic chemicals are involved.


    Cytochrome c release from mitochondria into the cytosol is determined by Western blotting using the cytochrome c antibody provided in the kit.


    The anti-Cytochrome c antibody is a mouse monoclonal antibody that reacts with denatured human, mouse, and rat cytochrome c.


    Cytochrome c release assay protocol summary:
    - collect cells, centrifuge and wash with PBS
    - resuspend in cytosol extraction buffer mix
    - homogenize cells with a dounce tissue grinder
    - centrifuge homogenate at 700 x g for 10 min
    - collect supernatant and centrifuge at 10,000 g for 30 min, collect supernatant as cytosolic fraction
    - resuspend pellet in mitochondrial extraction buffer mix and save as mitochondrial fraction
    - analyze cytosolic and mitochondrial fractions in western blotting with cytochrome c antibody

  • Notes

    This kit was previously called Cytochrome c Releasing Apoptosis Assay Kit.

    Cytochrome c plays an important role in apoptosis. The protein is located in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from the mitochondria into cytosol where it binds to Apaf-1. The cytochrome c/Apaf-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases.


    Other apoptosis assays

    For more apoptosis assays, review the apoptosis assay and apoptosis marker guide.

Properties

Images

  • Inhibition of cytochrome c release from mitochondria in SK-N-SH cells
    Inhibition of cytochrome c release from mitochondria in SK-N-SH cellsSun Q., PLoS One 9(6), Fig 4. doi: 10.1371/journal.pone.0098866. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Cytochrome C release was measured using Cytochrome C releasing apoptosis assay kit (ab65311). Blots showing immunoreactive bands for cytochrome c in cytosol (Image A). Data was expressed in fold-increase of cytochrome c compared to vehicle. Protein expression levels were normalized to β-actin (Figure B).  Blots (Image C) of immunoreactive bands for cytochrome C in mitochondria. Figure D shows a fold-increase of cytochrome C compared to vehicle. Protein expression levels were normalized to COX IV. 

  • Functional assays: Cytochrome c Releasing Apoptosis Assay Kit (ab65311)
    Functional assays: Cytochrome c Releasing Apoptosis Assay Kit (ab65311)

    5x10e7 Jurkat cells were cultured in the absence (1-2) or presence of 2 uM Camptothecin (CPT) (ab120115) for 24 hours (3-4) or with 10 uM CPT for 4 hours (5-6). 30 uL cytosolic (1, 3, 5) and mitochondrial (2, 4, 6) extracts were loaded onto the gel. Membranes were probed with anti-Cytochrome C Mouse MAb (ab65311) (dilution 1:200) followed by Goat Anti-Mouse IgG (HRP) (ab97040) (dilution 1:2000).

    Bands were detected at the prediced size of 12 kDa.

Protocols

References

This product has been referenced in:

  • Kawala RA  et al. Kenalog modified by ionizing radiation induces intrinsic apoptosis mediated by elevated levels of reactive oxygen species in melanoma cancer. Oncol Rep 41:1837-1850 (2019). Read more (PubMed: 30569155) »
  • Koutsogiannis Z  et al. G418 induces programmed cell death in Acanthamoeba through the elevation of intracellular calcium and cytochrome c translocation. Parasitol Res 118:641-651 (2019). Read more (PubMed: 30617503) »
See all 17 Publications for this product

Publishing research using ab65311? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 12 Abreviews or Q&A

Abreview for ab65311 Application: WB Sample species: Pig

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
Type: Cell lysate, mitochondrial fraction
Amount of protein used: 10 ug
Specification: Porcine kidney epithelial cells (PK-15)
Reduce/denaturing condition: Reduced, Denaturing, 4-20% gradient gel
Blocking buffer/concentration/time/temperature: Milk solution (+0.1% Tween-20)/5%/1 h/~20°C
Antibody dilution: 1:1000
Incubation time/temperature: 18 h/4°C
Diluent: 5% milk solution (+0.1% Tween-20)
Secondary antibody information: Non-Abcam antibody/Goat-anti-mouse/Polyclonal/HRP/1:5000
Detection method: ECL
Exposure: 10 mins
Observed band size: Specific: 12 kDa

Mr. Sam Hardy

Verified customer

Submitted Jan 18 2017

Question

On the step #9 of the booklet you write "Load 10 ug each of the cytosolic and mitochondrial fractions isolated from uninduced and induced cells on a 12% SDS-PAGE" and I have two questions:

1- Are the Cytosolic Fraction and Mitochondrial Fraction per se suitable for a BCA dosage to determine the volume necessary to obtain 10 ug.

2- Do we have to perform a denaturation step of the samples by heating at 95°C during 5 min as usual?

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Answer

1-

Are the Cytosolic Fraction and Mitochondrial Fraction per se suitable for a BCA dosage to determine the volume necessary to obtain 10 ug? -
Yes, a BCA assay can be used but it is better to use a reducing agent compatible assay.

2-
Do we have to perform a denaturation step of the samples by heating at 95°C during 5 min as usual? -
Yes, you heat with the loading buffer as you would for a western blot using SDS-PAGE

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Question

can the antibody be sold separately from the kit?

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Answer

The antibody from this kit is now available separately on our catalog as product ab189738 https://www.abcam.com/cytochrome-c-mouse-mab-ab189738.html.

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Abreview for ab65311 Application: WB Sample species: Human

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
Type: Tissue lysate
Amount of protein used: 35 ug
Specification: Heart
Reduce/denaturing condition: Reduced, Denaturing, 12% gel
Blocking buffer/concentration/time/temperature: BSA/5%/1 h/25oC
Antibody dilution: 1:1000
Incubation time/temperature: 18 h/4oC
Diluent: BSA
Secondary antibody information: Non-Abcam antibody/Donkey-anti-mouse/Polyclonal/HRP/1:10000
Detection method: ECL+
Exposure: 5 s
Observed band size: Specific: 12 kDa

Abcam user community

Verified customer

Submitted Jul 05 2013

Abreview for ab65311 Application: WB Sample species: Mouse

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
Type: Tissue lysate
Amount of protein used: 35 ug
Specification: Heart
Reduce/denaturing condition: Reduced, Denaturing, 12% gel
Blocking buffer/concentration/time/temperature: BSA/5%/1 h/25oC
Antibody dilution: 1:1000
Incubation time/temperature: 18 h/4oC
Diluent: BSA
Secondary antibody information: Non-Abcam antibody/Donkey-anti-mouse/Polyclonal/HRP/1:10000
Detection method: ECL+
Exposure: 5 s
Observed band size: Specific: 12 kDa

Abcam user community

Verified customer

Submitted Jul 05 2013

Question



Inquiry: In the Cytochrome c releaseing apoptosis assay kit (ab65311), How important is the homogenization step for cultured cells (it may be required for tissue). Can it be replaced with vortexing at high speed for say 10-15 sec. Cytosol extraction buffer, being hypotonic, will anyway break the cell membrane and release cytosol. thank you!

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Answer

Thank you for contacting us.

Yes, homogenization is a critical part of this protocol. You need to ensure that the cells have lysed well and released the cellular fractions in order to ensure that you see the relative difference in the levels of Cyt-C. So please do not use vortexing as an alternative to homogenization.

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

Phone call requesting information on whether tissue which has been frozen at -80 for a few days is likely to be suitable with the kit and if there is a positive control (i.e. with the control sample is he expected to see a band in WB or not)?

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Answer

Thank you for contacting us and your interest in our products.

As discussed over the phone I have looked further into your query in regards to the Cytochrome c Releasing Apoptosis Assay Kit (ab65311).

In reply to your questions, when using the kit you should be able to see the level of apoptosis as the ratio of Cytochrome C you see in normal vs apoptotic tissue in the mitochondiral vs the cytoplasmic fractions separated with the kit. Therefore both fractions will vouch for the protocol working fine. Only if you do not see any bands in any fraction it will indicate that the isolation process did not work out effectively.

We would recommend using a fresh sample with the kit. If you have to use frozen samples this may work with the kit but we have not tested it ourselves and therefore cannot guarantee its performance. We would suggest that you modify the protocol by washing the tissue with ice cold PBS and then resuspend each 10 mg of tissue in 1.0 ml of cytosol extraction buffer. Subsequently follow the protocol exactly as mentioned on the protocol from step 5.
I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

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Question

CLL primary cells, human
are the cytosolic and the mitochondrial extraction buffers detergent based or rather hypotonic buffers?
compositions?
is the nucleus part of the mitochondrial fraction?

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Answer

Thank you for contacting us.

The lab let me know the following:

The cytosolic and the mitochondrial extraction buffers detergent based. But the exact composition is proprietary.
The nucleus will be a part of the mitochondrial fraction.

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

Please can you let us know how to reconsitute the lyophilsed protease inhibitor cocktail that comes with ab65311.

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Answer

Thank you for your telephone enquiry yesterday.

I can confirm that to reconstitute the protease inhibitor cocktailprovided withab65311 Cytochrome c Releasing Apoptosis Assay Kit:

Add 250 ìl DMSO to dissolve the 500X Protease Inhibitor Cocktail before use.

I am sorry this information has regrettably not been provided on the protocol with thedatasheet on this occasion. I have put in a request to our datasheets team to make this addition.

I hope this information will be helpful. If you have any further questions, please do not hesitate to contact us.

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Question

Phone call enquiring about the difference between the Mitochondria/Cytosol Fractionation Kit (100 assay) (ab65320) and Cytochrome c Releasing Apoptosis Assay Kit (ab65311).

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Answer

Thank you for your interest in the Mitochondria/Cytosol Fractionation Kit (ab65320) and Cytochrome c Releasing Apoptosis Assay Kit (ab65311). Both kits are essentially the same in the preparation of the samples, however, following the separation of the Mitochondira and cytosol the Cytochrome c Releasing Apoptosis Assay Kit further allows you to determine Apoptosis based on the release of cytochrome C. This is performed through the application of the Anti-Cytochrome c Mouse monoclonal antibody supplied with this kit. I hope this information has been of help. If you need any further information please do let me know.

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1-10 of 12 Abreviews or Q&A

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