Overview

  • Product name
    Cytochrome c Profiling ELISA Kit
    See all Cytochrome c kits
  • Detection method
    Colorimetric
  • Sample type
    Cell culture extracts, Tissue
  • Assay type
    Sandwich (qualitative)
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    Abcam’s Cytochrome C in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the measurement of Cytochrome C in Human, mouse, rat and bovine samples of purified mitochondria, tissue and cell extracts.


    Abcam’s Cytochrome C ELISA kit is used to determine the amount of the cytochrome c in a sample. Cytochrome c is immunocaptured within the wells and the amount is determined by adding a cytochrome c specific antibody conjugated with horseradish peroxidase (HRP). This peroxidase changes the substrate from colorless to blue. This reaction takes place in a time dependent manner proportional to the amount of protein captured in the wells. The rate of development of blue color can be followed at 600 nm or the reaction can be stopped, at a user defined time, by the addition of 100 μL of 1.5 N HCl to each well (not supplied) and read at 450 nm.

  • Notes

    The permeabilization of the mitochondrial outer membrane and the consequent release of cytochrome c and other apoptogenic proteins from the mitochondrial intermembrane space into cytoplasm is considered hallmark of many apoptotic pathways leading to cell death. Therefore assaying these proteins in mitochondrial and cytoplasmic fractions is a prime interest of many researchers. Most of the current biochemical methods of quantification of cytoplasmic and mitochondrial cytochrome c involve mechanical disruption of cells to obtain mitochondria-enriched and cytosolic fractions. These methods are time-consuming and limited to small number of samples.

    This cytochrome c detection assay was developed in conjunction with Abcam’s ab109719 Cell Fractionation Kit - Standard and ab109718 Cell Fractionation Kit - HT, and the results were validated with ab110417 ApoTrack™ Cytochrome c Apoptosis Cocktail and with ab110415 ApoTrack™ Apoptosis Detection Kit for Immunocytochemistry.

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab110172 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent dilution.

Images

  • Principle of Sandwich ELISA used in Cytochrome c Protein Quantity Microplate Assay Kit (ab110172). Substrate plus HRP or AP conjugated to streptavidin provide a colorimetric signal that is readable by any plate readers capable of standard ELISA absorbance measurements.

Protocols

References

This product has been referenced in:
  • Giang AH  et al. Mitochondrial dysfunction and permeability transition in osteosarcoma cells showing the Warburg effect. J Biol Chem 288:33303-11 (2013). ELISA ; Human . Read more (PubMed: 24100035) »
  • Eliseev RA  et al. Cyclophilin D interacts with Bcl2 and exerts an anti-apoptotic effect. J Biol Chem 284:9692-9 (2009). Read more (PubMed: 19228691) »
See all 2 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Question
Answer

Thank you for contacting us.

The kits are guaranteed for 6 months if stored as recommended on the datasheet.

Please note there are components in the kits that are supposed to stored at different temperatures e.g. -20 or -80C, so we recommend carefully checking the datasheet and protocol booklet.

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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Abreviews
Performed cytochrome C ELISA to determine if any variation was occurring between spleen homogenates from different experimental animal groups. Followed kit according to manufacturers instructions. Protein in spleen homogenates measured using a BCA assay. Made up 100ul of sample in kit solution 1 at 1mg/ml as a starting point. After development solution added, performed kinetic measurements at 90 sec apart at 600nm on plate reader. Good linear development of each sample. Samples analysed in triplicate and had good reproducibility between wells. Blank wells showed little background labelling. Stopped reaction with HCl and also took a final reading. Analysed samples using difference in OD over time as recommended in instruction manual and gave good results. Overall a simple kit to use that gave reliable results quickly.

Ms. Laura McCulloch

Verified customer

Submitted Jan 29 2014

Abreviews
Performed cytochrome C ELISA to determine if any variation was occurring between spleen homogenates from different experimental animal groups. Followed kit according to manufacturers instructions. Protein in spleen homogenates measured using a BCA assay. Made up 100ul of sample in kit solution 1 at 1mg/ml as a starting point. After development solution added, performed kinetic measurements at 90 sec apart at 600nm on plate reader. Good linear development of each sample. Samples analysed in triplicate and had good reproducibility between wells. Blank wells showed little background labelling. Stopped reaction with HCl and also took a final reading. Analysed samples using difference in OD over time as recommended in instruction manual and gave good results. Overall a simple kit to use that gave reliable results quickly.

Ms. Laura McCulloch

Verified customer

Submitted Jan 23 2014

Answer

For the kit ab110172, you will need a spectrophotometer that can measure at 450nm. These are generally available in most labs, so perhaps a neighboring lab has one? I really don't have a recommendation of a specific one to use, just as long as it can measure 450nm in a 96 well plate format. I found one online by Googling here: http://www.biochrom.co.uk/product/37/biochrom-anthos-2020-microplate-reader.html

We have cytochrome C ELISA kits, but these also require a reader.
https://www.abcam.com/cytochrome-c-quantitative-elisa-ab154471.html
https://www.abcam.com/cytochrome-c-human-elisa-kit-with-color-giving-dyes-ab119521.html

Are you set up to do WB? We have a cytochrome c apoptosis WB antibody cocktail as ab110415.
https://www.abcam.com/apotrack™-cytochrome-c-apoptosis-wb-antibody-cocktail-ab110415.html

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Answer

Thank you for contacting us.

The Detergent supplied with MSA41 (ab110172) contains SDS. It will lyse all membranes including plasma membrane and mitochondrial membranes. Remember the purpose of this kit is to measure total cytochrome c concentration in whatever sample (subcellular fractions, cultured cells and tissues). The kit is compatible with two cell fractionation kits (ab109718 and ab109719). These two fractionation kits will generate cytosolic, mitochondrial and nuclear fractions and the cytochrome c can be assayed in these fractions by ab110172.

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

6 months at 4 degrees C is a reasonable length of storage.

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Answer

Thank you for contacting us.

The lab let me know that, if the sample is prepared according to the procedure in section C then:

All cytochrome c, regardless its subcellular location, is extracted and present in the supernatant. Pellet should not contain any cytochrome c.
I am not certainwhat the customer is refering to as negative and positive control, but as noted inchapter 7, section A, step 1: null-buffer control should be used in every assay (negative control).




In general, a negative control would not contain the protein of interest, while the positive control contains the protein of interest.

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 patience.

In Chapter 6, section C describes extraction of total protein with Detergent. The great majority of proteins, including all cytochrome c are solubilized by the Detergent. The centrifugation step (step 6) is to clear the sample of insoluble material. So the pellet should be discarded and the cytochrome c should be measured in the supernatant.

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 the following information from the lab so far:

This ELISA-based assaymeasures total cytochrome c in the sample of interest. These samples can be extracts of whole cultured cells and tissue homogenates, or alternatively cytosolic, mitochondrial and nuclear fractions prepared with the use of ab109719 / ab109718.

Depending on the sample type, two separate protocols are provided in ab110172 how to do final preparation of the sample of interest for the ELISA measurement, however ab110172 does not contain the protocol or reagents to fractionate the cells.

I am still waiting to hear back regarding step 6 on page 13 of the online protocol, and will let you know as soon as I have an answer for you.

I hope this information is already 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.
These kits will be OK to use with snap frozen tissues.
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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https://www.abcam.com/abreviews

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1-10 of 12 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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