Overview

  • Product name
    Anti-Cytochrome C antibody
    See all Cytochrome C primary antibodies
  • Description
    Sheep polyclonal to Cytochrome C
  • Host species
    Sheep
  • Specificity
    This antibody reacts specifically with cytochrome C (12.3 kDa).
  • Tested applications
    Suitable for: WB, IP, ICC/IF, ICCmore details
  • Species reactivity
    Reacts with: Rat, Rabbit, Dog, Human
  • Immunogen

    Full length native protein (purified) (Rabbit).

Applications

Our Abpromise guarantee covers the use of ab6400 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
WB
IP
ICC/IF
ICC
  • Application notes
    ICC: 1/650.
    IF: Use at an assay dependant dilution.
    IP: 1 µl will immunoprecipitate cytochrome C from 0.5 mg of MCF-7 cell lysate.
    WB: 1/10000 using MCF-7, Rat-1, MDCK and Jurkat cell lysates, anti-sheep IgG conjugated to peroxidase and ECL.

    Not tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    • Function
      Electron carrier protein. The oxidized form of the cytochrome c heme group can accept an electron from the heme group of the cytochrome c1 subunit of cytochrome reductase. Cytochrome c then transfers this electron to the cytochrome oxidase complex, the final protein carrier in the mitochondrial electron-transport chain.
      Plays a role in apoptosis. Suppression of the anti-apoptotic members or activation of the pro-apoptotic members of the Bcl-2 family leads to altered mitochondrial membrane permeability resulting in release of cytochrome c into the cytosol. Binding of cytochrome c to Apaf-1 triggers the activation of caspase-9, which then accelerates apoptosis by activating other caspases.
    • Involvement in disease
      Defects in CYCS are the cause of thrombocytopenia type 4 (THC4) [MIM:612004]; also known as autosomal dominant thrombocytopenia type 4. Thrombocytopenia is the presence of relatively few platelets in blood. THC4 is a non-syndromic form of thrombocytopenia. Clinical manifestations of thrombocytopenia are absent or mild. THC4 may be caused by dysregulated platelet formation.
    • Sequence similarities
      Belongs to the cytochrome c family.
    • Post-translational
      modifications
      Binds 1 heme group per subunit.
    • Cellular localization
      Mitochondrion matrix.
    • Information by UniProt
    • Database links
    • Alternative names
      • CYC antibody
      • CYC_HUMAN antibody
      • CYCS antibody
      • Cytochrome c antibody
      • Cytochrome c somatic antibody
      • HCS antibody
      • THC4 antibody
      see all

    References

    This product has been referenced in:
    • Ramonet D  et al. Optic atrophy 1 mediates mitochondria remodeling and dopaminergic neurodegeneration linked to complex I deficiency. Cell Death Differ : (2012). Read more (PubMed: 22858546) »
    • Melli G  et al. Spatially distinct and functionally independent mechanisms of axonal degeneration in a model of HIV-associated sensory neuropathy. Brain 129:1330-8 (2006). ICC/IF ; Rat . Read more (PubMed: 16537566) »
    See all 2 Publications for this product

    Customer reviews and Q&As

    1-10 of 12 Abreviews or Q&A

    Answer

    For a secondary to follow the primary anti-caspase 3 antibody, which is raised in rabbit, I suggest ab97051, which is goat anti-rabbit IgG, conjugated to HRP. This is assuming you are using an ECL-based detection after the secondary HRP conjugate. Since the primary is conjugated to biotin, you could also use an avidin-HRP conjugate or streptavidin-HRP conjugate instead of a secondary antibody, such as ab7403, Streptavidin-HRP. Here is a link to the product page: https://www.abcam.com/index.html?datasheet=7403 For your experiment, I think the biotin-streptavidin bond itself should not interfere with the antibody recognition site, but we have no data for what you propose. Regarding the ability of the antibody ab6400 to inhibit or neutralize cytochrome C, we have no data for this either. To our knowledge, the antibody has only been tested for western blotting, immunoprecipitation, and ICC/IF. I hope this helps somewhat. If you have any questions, please contact us.

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    Answer

    Thank you for contacting us. We do not have this antibody, ab6400, as a streptavidin conjugate and we do not offer conjugation service. However, we do have conjugation kits that make conjugation to avidin very simple. If you are interested, the catalogue numbers of the conjugation kits are ab102860, ab102861, and ab102862. They differ in the amount of antibody that can be conjugated. https://www.abcam.com/index.html?datasheet=102860 These kits require purified antibody to be effective. Antibody ab6400 is whole, unpurified serum, and is not suitable for conjugation. I recommend ab80911, an azide- and BSA-free mouse monoclonal antibody. However, it has only been tested in a few applications: immunoprecipitation, ELISA, and flow cytometry, so it may not be suitable for your needs. https://www.abcam.com/index.html?datasheet=80911 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 your enquiry. To clarify I can tell you that this antibody has tested on on fixed cells, not frozen tissue. A MDCK (Dog kidney) lysate was tested by western. However, canine cells are yet to be tested by immunocytochemistry. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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    Answer

    Thank you for your enquiry. We do have a sheep polyclonal to cytochrome C, (ab6400) which has been tested in dog. This is raised against the whole protein, so I am sorry I cannot be more specific about the epitopes. I have found a website and a reference which may be useful to you. The website seems to indicate that subunit II of the protein is transmembrane. http://www.ebi.ac.uk/interpro/DisplayIproEntry?ac=IPR011759 Order of Steps in the Cytochrome c Folding Pathway: Evidence for a Sequential Stabilisation Mechanism Journal of Molecular Biology, Volume 359, Issue 5, 23 June 2006, Pages 1410-1419 Mallela M.G. Krishna, Haripada Maity, Jon N. Rumbley, Yan Lin and S. Walter Englander I hope this helps. Please get back to me if you require any more information, and I will be happy to help. With thanks

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    Question

    BATCH NUMBER 71313 ORDER NUMBER 63466 DESCRIPTION OF THE PROBLEM This sample is non-specific stained. So we tested a secondary antibody. But, secondary antibody wasn?t a problem. SAMPLE Species : Human Cell line : A549 Cell Type : Cancer cell PRIMARY ANTIBODY Manufacturer : abcam Species : rabbit, cross-react : dog, Human, rat Diluent ; used at assay an dependent diluents. Dilution : 500:1 ~ 50:1 Incubation time : 2hr~ overnight Wash step : With PBS containing 0.02% tween 20 and 1% BSA for 10min (3times) SECONDARY ANTIBODY Manufacturer : abcam Species : sheep Diluent ; used at assay an dependent diluent. Dilution : 1000:1 ~ 200:1 Incubation time : 1hr Wash step : With PBS containing 0.02% tween 20 and 1% BSA for 10min (3times) DETECTION METHOD Immunofluorescence method fluorescence Microscope POSITIVE AND NEGATIVE CONTROLS USED Used to other company samples. We always use many antibodies by manufactured Phamingen. ANTIBODY STORAGE CONDITIONS Stored at -20?C . FIXATION OF SAMPLE (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.) We fixated to paraformaldehyde 4% at Room Temperature, 1 hr. PERMEABILIZATION STEP 1. 0.1% NP40 or 1% triton X-100 in PBS ? 1% BSA incubation at RT 30min. 2. Washing for 1X PBS (3 times) BLOCKING CONDITIONS Buffer : PBS containing 0.02% tween 20 and 1% BSA Time period : washed the cells with PBS and buffer 10min (3 times) Blocking agent : 1% BSA agent HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? 1. Cell fixation 2. Permeabilization 3. added to primary antibody 4. added to secondary antibody 5. mounting 6. observation We tried to many methods for IF. But, we gave to be a same result.

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    Answer

    I am sorry you are having problems with ab6400. Thank you for taking the time to fill in our online questionnaire, it is very useful. I would like to recommend a shorter fixation time (30min) or try ice cold methanol fixation and to follow the recommended protocol below(in particular incubating the primary and secondary antibodies in PBST): 1. Draw off culture media and rinse the cells 2 X with warm PBS. 2. Transfer coverslips to 6 well culture dishes, one coverslip per well. It is recommended to line the bottom of the wells with parafilm for easier handling. 3. Add 30 µl fixative (4% paraformaldehyde) for 30 min at room temperature. 4. Wash the coverslips with PBS, 1 X for 5 mins with gentle agitation. Do not shake cells. 5. Add 30 µl PBS containing 1% Triton-X-100 and incubate for 10 min at room temperature. 6. Wash cells with PBS containing 0.02% Tween 20 for 5 min. 7. Wash cells with PBS containing 0.02% Tween 20 and 1% BSA for 5 min. 8. Incubate the coverslips with 30 µl anti-Cytochrome C diluted approximately 1:650 (1.5 µl/ml) in PBS containing 3% BSA and incubate in a humidified chamber 45 min 37°C. 9. Wash the cells with PBS containing 0.02% Tween 20 and 1% BSA for 5 min. 10. Add 30 µl of a 1:40 dilution of anti-sheep IgG conjugated with FITC in PBS containing 3% BSA and incubate in a humidified chamber 45 min 37°C. 11. Wash the cells with PBS containing 0.02% Tween 20 and 1% BSA for 5 min. 12. Wash the cells with PBS for 5 min. 13. Dry in a 37°C oven (bacterial incubator) for 45 min. 14. Use 10-15 µl mounting media to mount each coverslip onto a slide. Add mounting media on side, and add coverslip with cells facing mounting media on top. Seal with clear nail polish and label slides. 15. Examine the cells under a fluorescent microscope. The positive controls recommended are MCF-7, Rat-1, MDCK and Jurkat cells I hope these suggestions will help, please do not hesitate to contact us if you have further problems and we will arrange a replacement antibody to be sent to you,

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    Answer

    All the information we have on species cross reactivity is specified on the datasheet. To our knowledge, cross reactivity with Xenopus has not yet been tested. We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement. Should you decide to test an antibody in an application for which we do not have any information, please let us know how you get on and in return we will forward a GBP10/ USD15/ EUR15 Amazon gift voucher

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    Answer

    This antibody is supplied as whole serum and is recommended for use in WB at a dilution of 1:10,000 with whole cell lysates. Are you using whole cell extracts - from what species is your cytochrome C derived? You say that you have used the anitbody at 1:2000 dilution which gave almost no band. I suggest that you try 1:500 and 1:200 dilutions. Please keep in touch and let me know how you get on. Danielle N. Miller PhD Head of Customer Services

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    Question
    Answer

    No, I am afraid that we do not sell secondary antibodies conjugated to gold particles

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    Question
    Answer

    To our knowledge this antibody has not been tested on paraffin embedded specimens.

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    Question
    Answer

    This antibody has not been tested on murine cytochrome c, but given the conservation of this protein, we would expect it to work for mouse.

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    1-10 of 12 Abreviews or Q&A

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