Overview

  • Product name
    Anti-Caspase-9 antibody [E23]
    See all Caspase-9 primary antibodies
  • Description
    Rabbit monoclonal [E23] to Caspase-9
  • Host species
    Rabbit
  • Specificity
    This antibody should recognise both the pro-[40kDa] form and p35 cleaved form of Caspase-9.
  • Tested applications
    Suitable for: WB, IHC-P, Flow Cyt, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human Caspase-9 aa 250-350. The exact sequence is proprietary.
    Database link: P55211

  • Positive control
    • WB: Jurkat and HeLa cell lysates. IHC-P: Human skeletal muscle and cervical carcinoma tissues. ICC/IF: HepG2 cells. Flow Cyt: K562 cells. IP: HeLa whole cell lysate.
  • General notes

    A trial size is available to purchase for this antibody.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab32539 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 1/1000 - 1/10000. Predicted molecular weight: 46 kDa.

We recommend overnight incubation at 4°C.

IHC-P 1/50 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Flow Cyt 1/100 - 1/250.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP 1/80 - 1/100.
ICC/IF 1/500.

For unpurified use at 1/50.

Target

  • Function
    Involved in the activation cascade of caspases responsible for apoptosis execution. Binding of caspase-9 to Apaf-1 leads to activation of the protease which then cleaves and activates caspase-3. Proteolytically cleaves poly(ADP-ribose) polymerase (PARP).
    Isoform 2 lacks activity is an dominant-negative inhibitor of caspase-9.
  • Tissue specificity
    Ubiquitous, with highest expression in the heart, moderate expression in liver, skeletal muscle, and pancreas. Low levels in all other tissues. Within the heart, specifically expressed in myocytes.
  • Sequence similarities
    Belongs to the peptidase C14A family.
    Contains 1 CARD domain.
  • Developmental stage
    Expressed at low levels in fetal heart, at moderate levels in neonate heart, and at high levels in adult heart.
  • Post-translational
    modifications
    Cleavages at Asp-315 by granzyme B and at Asp-330 by caspase-3 generate the two active subunits. Caspase-8 and -10 can also be involved in these processing events.
  • Information by UniProt
  • Database links
  • Alternative names
    • APAF-3 antibody
    • APAF3 antibody
    • Apoptosis related cysteine peptidase antibody
    • Apoptotic protease Mch-6 antibody
    • Apoptotic protease-activating factor 3 antibody
    • CASP-9 antibody
    • CASP9 antibody
    • CASP9_HUMAN antibody
    • Caspase 9 apoptosis related cysteine peptidase antibody
    • Caspase 9 Dominant Negative antibody
    • Caspase 9c antibody
    • Caspase-9 antibody
    • Caspase-9 subunit p10 antibody
    • ICE LAP6 antibody
    • ICE like apoptotic protease 6 antibody
    • ICE-LAP6 antibody
    • ICE-like apoptotic protease 6 antibody
    • MCH6 antibody
    • PPP1R56 antibody
    • protein phosphatase 1, regulatory subunit 56 antibody
    • RNCASP9 antibody
    see all

Images

  • Lanes:
    Lane 1: Wild-type HAP1 whole cell lysate (40 µg)
    Lane 2: CASP9 knockout HAP1 whole cell lysate (40 µg)
    Lane 3: HeLa whole cell lysate (40 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab32539 observed at 45 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab32539 was shown to specifically recognize Caspase-9 in wild type HAP1 cells along with additional cross-reactive bands. No band was observed when Caspase-9 knockout samples were examined. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. Ab32539 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue labelling Caspase-9 with purified ab32539 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Caspase-9 with purified ab32539 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Flow Cytometry analysis of K562 cells labelling Caspase-9 with purified ab32539 at 1/250 (red). Cells were fixed with 100% methanol. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • ab32539 (purified) at 1/80 immunoprecipitating Caspase-9 in HeLa whole cell lysate.

    Lane 1 (input): HeLa whole cell lysate (10µg)

    Lane 2 (+): ab32539 + HeLa whole cell lysate.

    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32539 in HeLa whole cell lysate.

    For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • All lanes : Anti-Caspase-9 antibody [E23] (ab32539) at 1/1000 dilution (purified)

    Lane 1 : HeLa whole cell lysate - treated with Camptothecin
    Lane 2 : HeLa whole cell lysate - untreated

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

    Predicted band size: 46 kDa
    Observed band size: 35,46 kDa why is the actual band size different from the predicted?



    Blocking and dilution buffer: 5% NFDM/TBST.

  • Overlay histogram showing K562 cells stained with unpurified ab32539 (red line). The cells were fixed with 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 (unpurified ab32539, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was anti-rabbit DyLight® 488 (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in K562 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Caspase 9 with unpurified ab32539 at a dilution of 1/50.

  • All lanes : Anti-Caspase-9 antibody [E23] (ab32539) at 1/20000 dilution (unpurified)

    Lane 1 : Jurkat cell lysate - untreated
    Lane 2 : Jurkat cell lysate - treated with Camptothecin

    Predicted band size: 46 kDa
    Observed band size: 48 kDa why is the actual band size different from the predicted?
    Additional bands at: 28 kDa. We are unsure as to the identity of these extra bands.

References

This product has been referenced in:
  • Yan H  et al. NR4A1-induced increase in the sensitivity of a human gastric cancer line to TNFa-mediated apoptosis is associated with the inhibition of JNK/Parkin-dependent mitophagy. Int J Oncol 52:367-378 (2018). Read more (PubMed: 29207128) »
  • Jin Q  et al. DUSP1 alleviates cardiac ischemia/reperfusion injury by suppressing the Mff-required mitochondrial fission and Bnip3-related mitophagy via the JNK pathways. Redox Biol 14:576-587 (2018). Read more (PubMed: 29149759) »
See all 54 Publications for this product

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Tissue lysate - whole (liver)
Specification
liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Dec 22 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Fadu)
Loading amount
30 µg
Specification
Fadu
Treatment
RT8gray for 48hrs+Metformin400mM
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Apr 24 2013

Answer


Full length caspase-9 will cleave into two units, caspase-9 p35 subunit and caspase-9 p10 subunit. The immunogen of ab32539 is a synthetic peptide corresponding to the C terminus of caspase-9 p35 subunit. Therefore, the antibody is able to detect both full length caspase-9 and cleaved caspase-9 p35 subunit.

Read More

Answer

Thank you for your inquiry.
I am happy to confirm that all these antibodies are tested and guaranteed to work with human samples.
You are correct that we do not have any reports yet about the use of these antibodies in muscle. I assume that is your customers area of research. It is not possible for us to test our product with all available tissues and therefore I would like to re-assure you and your customer that these antibodies are covered by our Abpromise guarantee when positive controls beside muscle are used in experiments.
I would recommend doing this anyway especially if your customer does not know what to expect in muscle as it is part of a research project.
Maybe your customer would like to consider writing an Abreview after they used the antibodies in their experiments? They would earn Abpoints that can be redeemed a discount on future purchases or Amazon voucher.
I hope this information is helpful and wish your customer good luck in their experiments.

Read More

Answer

La référence ab2014 n'a pas encore été testée chez le rat, c'est pour cela que j'ai préféré comparer les séquences.

Nous serions ravis si vous pouviez nous communiquer vos résultats. Ceci nous permettra peut-être et je l'espère d'ajouter le rat aux espèces validées pour cet anticorps.

J'ai donc procédé à l'envoi d'une unité de ab2014 en remplacement du ab32539. Le numéro de la commande est : ******.

Read More

Answer

Merci pour votre réponse.

L'anticorps ab63488 est en effet garanti pour fonctionner en WB et IHC-P. Je suis d'accord avec vous, l'image du blotn'est pas très attractive.

Une autre référence qui a retenu mon attentionest ab2014, un polyclonal de lapin, testé WB et ICH-P et dont l'immunogène présente 100%d'homologie avec la protéine derat. J'orienteraispersonnellement mon choixpour cet anticorps, https://www.abcam.com/ab2014

Merci de m'indiquer ce que vous en pensez.

Read More

Answer

Merci de nous avoir contactés de nouveau.

Je suis vraimentdésolé d'apprendre que le second lot de ab32359 ne fonctionne pas toujours pas comme attendu.

Afin de solutionnerce problème, je peux vous proposer l'envoi gratuit (puisque le produit est toujours sous garantie) de :
- soit un autre lot de ab32359 (différent des 2 lots précédemment reçus),
- soit une unité de ab25758, https://www.abcam.com/ab25758
- soit une unité de ab63488, https://www.abcam.com/ab63488
- soit une unité de ab115161, https://www.abcam.com/ab115161
- soit une unité de ab2013, https://www.abcam.com/ab2013
- soit une unité de ab47537, https://www.abcam.com/ab47537

Tous les anticorps anti-caspase9listés ci-dessus sont donnés pour fonctionner en Western Blot et sur échantillons de rat.

Merci de m'indiquer la résolution qui vous conviendrait le mieux. Bien entendu,l'éditiond'un remboursement oud'un avoir estégalement possible.

Je vous fais parvenir dans un autre email un devis pour les anticorps ab62484 et ab47131.

Read More

Answer

As far as we know, rabbits do not have specific IgG subclasses like mice or
humans. All of our antibodies are simply considered to be rabbit IgG.
I hope this is helpful. Please contact me again if you have any further questions.

Read More

Answer

Je suis vraiment désolé d'apprendre que vous rencontrez un autre problème avec cet anticorps. Pourriez-vous s'il vous plait tester cet anticorps. Si il fonctionne je vous enverrai une nouvelle unité en prenant soin de faire vérifier le tube avant expédition. Si il ne fonctionne pas nous essaierons de trouver ensemble une résolution scientifique ou commerciale. Je recommanderais une dilutiuon au 1/1000 pour cet anticorps. Dans l'attente de vos résultats, je vous souhaite une bonne fin de semaine ainsi qu'un bon week-end.

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Question
Answer

Merci de nous avoir contactés. Nous sommes désolés que le produit ab32539 que vous avez reçu ne fonctionne pas comme attendu. Comme convenu j'ai mis en place l'envoi d'une unité de ab32539 d'un nouveau lot. Le numéro de commande de remplacement gratuit de ce produit est : 994237. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition. Je me renseigne également pour l'immunogène du ab63342 afin de déterminer si il y a de bonnes chances qu'il fonctionne chez le rat ou non. N'hésitez pas à nous contacter lors d'une prochaine occasion.

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1-10 of 14 Abreviews or Q&A

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