Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E23] to Caspase-9
- Suitable for: WB, IHC-P, Flow Cyt, IP, ICC/IF
- Knockout validated
- Reacts with: Human
Product nameAnti-Caspase-9 antibody [E23]
See all Caspase-9 primary antibodies
DescriptionRabbit monoclonal [E23] to Caspase-9
SpecificityThis antibody should recognise both the pro-[40kDa] form and p35 cleaved form of Caspase-9.
Tested applicationsSuitable for: WB, IHC-P, Flow Cyt, IP, ICC/IFmore details
Species reactivityReacts with: Human
Synthetic peptide within Human Caspase-9 aa 250-350. The exact sequence is proprietary.
Database link: P55211
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
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.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
Storage bufferpH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
PurityProtein A purified
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
- Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
- Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
- Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
- Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
- Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
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.
|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.|
|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.|
For unpurified use at 1/50.
FunctionInvolved 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 specificityUbiquitous, 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 similaritiesBelongs to the peptidase C14A family.
Contains 1 CARD domain.
Developmental stageExpressed at low levels in fetal heart, at moderate levels in neonate heart, and at high levels in adult heart.
modificationsCleavages 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
- APAF-3 antibody
- APAF3 antibody
- Apoptosis related cysteine peptidase antibody
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).
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.
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.
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab32539 has been referenced in 119 publications.
- Sun X et al. Effect of Loureirin B on Crohn's disease rat model induced by TNBS via IL-6/STAT3/NF-?B signaling pathway. Chin Med 15:2 (2020). PubMed: 31911815
- Duan C et al. Drp1 regulates mitochondrial dysfunction and dysregulated metabolism in ischemic injury via Clec16a-, BAX-, and GSH- pathways. Cell Death Dis 11:251 (2020). PubMed: 32312970
- Zhao J et al. Long non-coding RNA ANRIL down-regulates microRNA-7 to protect human trabecular meshwork cells in an experimental model for glaucoma. Eur Rev Med Pharmacol Sci 23:3173-3182 (2019). PubMed: 31081068
- Amin AH et al. Ameliorative effects of melatonin against solid Ehrlich carcinoma progression in female mice. J Pineal Res 67:e12585 (2019). PubMed: 31066091
- Cui J et al. WITHDRAWN: Skullcapflavone I has a potent anti-pancreatic cancer activity by targeting miR-23a. Life Sci N/A:N/A (2019). PubMed: 31085241