Overview

  • Product name
    Anti-COX IV antibody [20E8C12]
    See all COX IV primary antibodies
  • Description
    Mouse monoclonal [20E8C12] to COX IV
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, IHC-FoFr, WB, ICC/IF, IHC-Fr, IHC-P, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Cow, Human, Pig, Drosophila melanogaster, Zebrafish
  • Immunogen

    Tissue, cells or virus corresponding to Cow COX IV.

  • Positive control
    • Human, bovine, murine and rat heart mitochondria.
  • General notes

    This antibody clone is manufactured by Abcam.

    This antibody makes an effective loading control for mitochondria. COXIV is generally expressed at a consistent high level. However, be aware that many proteins run at the same 16kD size as COXIV - our VDAC1 / Porin antibody makes a good alternative mitochondrial loading control for proteins of this size. Some caution is required when using this antibody as a loading control as COXIV expression can vary under some manipulations. An alternative mitochondrial loading control is Rabbit polyclonal to COX IV antibody (ab16056).

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab14744 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
Flow Cyt Use 0.5µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

IHC-FoFr Use at an assay dependent concentration. PubMed: 18698340
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa.
ICC/IF Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IP Use at an assay dependent concentration.

Target

  • Function
    This protein is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport.
  • Tissue specificity
    Ubiquitous.
  • Sequence similarities
    Belongs to the cytochrome c oxidase IV family.
  • Cellular localization
    Mitochondrion inner membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • AL024441 antibody
    • COX 4 antibody
    • COX IV 1 antibody
    • COX IV antibody
    • COX IV-1 antibody
    • Cox4 antibody
    • COX41_HUMAN antibody
    • Cox4a antibody
    • COX4B antibody
    • COX4I1 antibody
    • COX4I2 antibody
    • COX4L2 antibody
    • COXIV antibody
    • Cytochrome c oxidase polypeptide IV antibody
    • Cytochrome c oxidase subunit 4 isoform 1 mitochondrial antibody
    • Cytochrome c oxidase subunit 4 isoform 1, mitochondrial antibody
    • Cytochrome C Oxidase subunit IV antibody
    • Cytochrome c oxidase subunit IV isoform 1 antibody
    • Cytochrome c oxidase subunit IV isoform 2 (lung) antibody
    • Cytochrome c oxydase subunit 4 antibody
    • dJ857M17.2 antibody
    • MGC105470 antibody
    • MGC72016 antibody
    see all

Images

  • Anti-COX IV antibody [20E8C12] (ab14744) at 1/1000 dilution + Fruit fly (Drosophila melanogaster) heart tube tissue lysate at 10 µg

    Secondary
    HRP-conjugate Goat anti-mouse IgG polyclonal at 1/4000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 1 minute

    See Abreview

  • ab14744 at 1/100 staining MCF-10A cells (human mammary epithelial cell line) by ICC/IF. The cells were methanol/acetone fixed at -20C for 5 minutes, blocked with BSA and then incubated with the antibody for 16 hours. The positive tissue was colocalised with a mitotracker (mitotrackers are a series of patented mt-selective stains that are concentrated by active mt and well retained during cell fixation). An Alexa-Fluor ® 488 conjugated goat anti-mouse antibody was used as the secondary. The image shows COXIV staining in green and DAPI staining in blue.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab14744 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab14744, 0.5µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1](ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • All lanes : Anti-COX IV antibody [20E8C12] (ab14744) at 1/5000 dilution

    Lane 1 : Cytoplasmic fraction mouse NIH/3T3 cell lysate
    Lane 2 : Nuclear fraction mouse NIH/3T3 cell lysate

    Secondary
    All lanes : HRP conjugated goat anti-mouse antibody

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 16 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    This image is courtesy of an Abreview submitted by Camilla Skjerpen on 4 July 2005.

    See Abreview

  • ab14744 staining COX IV in rat brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Rats were anesthetized and intracardially perfused with 500 ml of normal saline at room temperature, followed by 500 ml of ice-cold, freshly made 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4). A Cy3 conjugated anti mouse antibody was used as secondary. 

  • All lanes : Anti-COX IV antibody [20E8C12] (ab14744) at 1 µg/ml

    Lane 1 : Isolated mitochondria from human heart at 5 µg
    Lane 2 : Isolated mitochondria from bovine heart at 1 µg
    Lane 3 : Isolated mitochondria from rat heart at 10 µg
    Lane 4 : Isolated mitochondria from murine heart at 10 µg

    Observed band size: 16 kDa why is the actual band size different from the predicted?

References

This product has been referenced in:
See all 187 Publications for this product

Customer reviews and Q&As

1-10 of 24 Q&A

Answer

Vielen Dank für Ihren Anruf.

Ich freue mich Ihnen bestätigen zu können, dass die Klone dieselben sind und früher bei MitoScience nur abgekürzt wurden.

Ich kann Ihnen auch bestätigen, dass eine Lagerung bei -20C korrekt ist solange die Antikörper lyophilisiert sind.

Bitte lagern sie die Antikörper bei +4C nach Rekonstitution.

Read More

Question
Answer

We would recommend the use of one of the following products for a loading control with zebrafish samples: AB15224 anti alpha Tubulin (https://www.abcam.com/alpha-tubulin-antibody-ab15246.html). This product has been featured in the following publication for use in WB on zebrafish (PubMed ID: 22581286). AB6046 anti beta Tubulin. This product has an excellent Abreview provided by a customer using in WB on zebrafish. This review is located at the following web address: https://www.abcam.com/beta-Tubulin-antibody-Loading-Control-ab6046-reviews.html?intabreviewid=15893 AB14744 anti- COX IV antibody (https://www.abcam.com/COX-IV-antibody-20E8C12-Mitochondrial-Loading-Control-ab14744-references.html). Another guaranteed product for use as loading control this product has been used in publication (PubMed ID: 18765295).

Read More

Answer

Thank you very much for your reply.

The common nuclear outer membrane proteins are nesprins, NAV3, and OPRS1. The nesprins and OPRS1 can also be found in the inner membrane or other organelles, but the NAV3 seems to be more specific to the outer membrane. Unfortunately we do not have any monoclonal antibodies to this target, just polyclonals such as ab101792-

https://www.abcam.com/nav3-antibody-n-terminal-ab101792.html

We do have monoclonal antibodies to nesprins, such as ab125931-

https://www.abcam.com/nesprin-1-antibody-mannes1a7a12-ab125931.html

Would either of these work for you?

I am sorry that we don't have more options at this time, but please let me know if there is anything else that I can help find for you, and I'll be happy to help.

Read More

Answer

Thank you very much for your enquiry.

I am sorry to hear that the nesprin and TOMM22 antibodies are not working as they should be. Could you tell me the catalog numbers of each of these antibodies, and also a description of the protocol that you're using? We do fully guarantee all of our products to work as stated on the datasheets, so I'd be happy to send replacements if applicable. Could you also send your original order or PO number?

If you'd like to continue probing for nesprin and TOMM22, we do have a couple of antibodies that work in Western blot with human and mouse samples-

https://www.abcam.com/TOMM22-antibody-ab57523.html

https://www.abcam.com/Nesprin3-antibody-ab74261.html

We also have several other antibodies that are targeted against mitochondrial or nuclear membrane-bound proteins that work in Western blot with mouse and human samples:

Mitochondrial

Prohibition antibody: https://www.abcam.com/Prohibitin-antibody-Mitochondrial-Marker-ab28172.html

MTCO1 antibody: https://www.abcam.com/MTCO1-antibody-1D6E1A8-Mitochondrial-Marker-ab14705.html

Nuclear

Lamin B1 antibodies: https://www.abcam.com/Lamin-B1-antibody-Nuclear-Envelope-Marker-ab16048.html

https://www.abcam.com/Lamin-B1-antibody-119D5-F1-Nuclear-Envelope-Marker-ab8982.html

Would one of these work for you?

I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that we can do for you.

Read More

Answer

Thank you very much for contacting us with your questions.

Will you be trying to isolate intact nuclei from mammalian cells? I'm not sure if there is an antibody that would be good for this, butthecell fractionation kit ab109719 that I mentioned beforemight be suitable. I'll need to check with the lab whether the nucleiu will still be intact using this kit, if that is your end goal.

To check the efficiency of a nuclear fractionation, I would recommend probing for either lamin B1 (66 kDa) or TATA binding protein (38 kDa). Both of these proteins will run higher than the histones but should be far enough away from the IgG heavy and light chains around 25 and 50 kDa. For mitochondria, I'd recommend a VDCA1/porin antibody (31 kDa), though this may be too close to the 25 kDa light chain band. You could also probe for COXIV(16 kDa) but this protein may be smaller than what you'd like. What species will your samples be from? I can find specific antibodies that are known to react with this species.

I'm not sure if you would be interested inthis, but we do have secondary antibodies that only bind to native primary antibodies so that the denatured chains won't be visible at 25 and 50 kDa following an IP. For anti-mouse IgG, we have ab131368-

https://www.abcam.com/mouse-IgG-VeriBlot-for-IP-secondary-antibody-HRP-ab131368.html

For anti-rabbit IgG, we have ab131366-

https://www.abcam.com/rabbit-IgG-VeriBlot-for-IP-secondary-antibody-HRP-ab131366.html

I look forward to hearing from you. Please let me know if you have any further questions or if there is anything else that we can do for you.

Read More

Answer

Unfortunately, we do not have a special "rat liver mitochondria loading control"but we have one antibody which may interest you:

ab16056 Anti-COX IV antibody - Mitochondrial Loading Control

https://www.abcam.com/index.html?datasheet=16056 (or use the following: https://www.abcam.com/index.html?datasheet=16056).

This antibody has two very positive Abreviews in WB and rat, and has been demonstrated to work in liver, however, the latter wasin IHC and human. Nonetheless, I think this is the right one for you in this case.

Read More

Answer

Thank you for your call yesterday and for your patience while I have been in touch with the lab scientists regarding your enquiry.

My contact at the lab recommends analyzing mitochondrial rupture by comparing the amount of cytochrome C in the cytoplasmic fraction versus the mitochondrial fraction. If the mitochondria are intact, there should be no cytochrome C in the cytoplasm. We havethe cell fractionation kit (ab109719) in conjunction with our antibody cocktail ab110415, which can be used for this purpose:

https://www.abcam.com/Cell-Fractionation-Kit-Standard-ab109719.html

https://www.abcam.com/apotrack™-cytochrome-c-apoptosis-wb-antibody-cocktail-ab110415.html

I've looked through some literature to see if there is amore traditional marker than can be used to distinguish intact and ruptured mitochondria, but unfortunately I couldn't find a reliable marker to recommend. Many researchers seem to rely on mitochondrial activity assays or staining patterns to distinguish the two, but in Western blot the cytochrome C study may be the most accurate.

I am sorry that I don't have more information, but I hope this will be useful. If you have any further questions or if there is anything else that we can do for you, please let me know and I'll be happy to help.

Read More

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with ab14744. The order number is XXXXX. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

Read More

Answer

Thank you for your enquiry.

Here is the link we discussed, to our ICC protocol:
https://www.abcam.com/index.html?pageconfig=resource&rid=11417

I hope this is helpful. Please contact me again if you have any further questions.

Read More

Question
Answer

Thank you for your call today and for your enquiry.

As we discussed, I would recommend either ab14744 or ab15895 for immunostaining of mitochondria in mouse cells-

https://www.abcam.com/COX-IV-antibody-20E8C12-Mitochondrial-Loading-Control-ab14744.html

https://www.abcam.com/VDAC1-Porin-antibody-Mitochondrial-Loading-Control-ab15895.html

We additionally have a CytoPainter mitochondrial dye, ab112143, that would be suitable to visualize mitochondria in live cells-

https://www.abcam.com/CytoPainter-Mitochondrial-Staining-Kit-Green-Fluorescence-ab112143.html

I hope this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

Read More

1-10 of 24 Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
For licensing inquiries, please contact partnerships@abcam.com

Sign up