Recombinant Anti-Caspase-9 antibody [EPR18107] (ab202068)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR18107] to Caspase-9
- Suitable for: IHC-P, WB, ICC/IF, IP
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-Caspase-9 antibody [EPR18107]
See all Caspase-9 primary antibodies -
Description
Rabbit monoclonal [EPR18107] to Caspase-9 -
Host species
Rabbit -
Tested applications
Suitable for: IHC-P, WB, ICC/IF, IPmore details -
Species reactivity
Reacts with: Mouse, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HeLa and C2C12 whole cell lysates; Human fetal brain, fetal heart, fetal kidney and fetal liver lysates. IHC-P: Human cervix carcinoma tissue. ICC/IF: HeLa cells. IP: HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.
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General notes
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.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR18107 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Assay kits
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Compatible Secondaries
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Isotype control
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab202068 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 |
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IHC-P |
1/300. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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WB | (3) |
1/2000. Detects a band of approximately 46, 35, 37 kDa (predicted molecular weight: 46 kDa).
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ICC/IF |
1/500.
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IP |
1/80.
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Notes |
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IHC-P
1/300. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
WB
1/2000. Detects a band of approximately 46, 35, 37 kDa (predicted molecular weight: 46 kDa). |
ICC/IF
1/500. |
IP
1/80. |
Target
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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
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
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Database links
- Entrez Gene: 842 Human
- Entrez Gene: 12371 Mouse
- Omim: 602234 Human
- SwissProt: P55211 Human
- SwissProt: Q8C3Q9 Mouse
- Unigene: 329502 Human
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Alternative names
- APAF-3 antibody
- APAF3 antibody
- Apoptosis related cysteine peptidase antibody
see all
Images
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All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/2000 dilution
Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : CASP9 knockout THP-1 cell lysate
Lane 3 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 46 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-Caspase-9 antibody [EPR18107] staining at 1/2000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab202068 was shown to bind specifically to Caspase-9. A band was observed at 45 kDa in wild-type THP-1 cell lysates with no signal observed at this size in CASP9 knockout cell line ab276122 (knockout cell lysate ab284219). To generate this image, wild-type and CASP9 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Caspase-9 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab202068 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab202068 was shown to recognize Caspase-9 when Caspase-9 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. ab202068 and ab8245 (loading control to GAPDH) were diluted at 1/2000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed withGoat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging. -
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Caspase-9 with ab202068 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).
Confocal image showing cytoplasmic and nuclear staining on HeLa cell line. The expression increased after treatment with staurosporine (1uM) for 4 hours.
The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab202067 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [EPR18107] (ab202068)
Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling Caspase-9 with ab202068 at 1/300 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.
Cytoplasmic and nuclear staining on Human cervix carcinoma tissue is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Caspase-9 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green).Green - target observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
This western blot image is a comparison between ab202068 and a competitor's top cited rabbit polyclonal antibody.
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All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/10000 dilution
Lane 1 : Untreated C2C12 (Mouse myoblast cell line) whole cell lysate
Lane 2 : C2C12 (Mouse myoblast cell line) treated with staurosporine 1uM for 4 hours whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution
Predicted band size: 46 kDa
Observed band size: 37,39,46 kDa why is the actual band size different from the predicted?
Exposure time: 3 minutesBlocking/Dilution buffer: 5% NFDM/TBST.
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All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/2000 dilution
Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 46 kDa
Observed band size: 46 kDa
Exposure time: 3 minutesBlocking/Dilution buffer: 5% NFDM/TBST.
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All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/2000 dilution
Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal liver lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 46 kDa
Observed band size: 46 kDa
Exposure time: 30 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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Caspase-9 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) treated with staurosporine 1uM for 4 hours whole cell lysate with ab202068 at 1/80 dilution.
Western blot was performed from the immunoprecipitate using ab202068 at 1/1000 dilution.
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.
Lane 1: HeLa treated with staurosporine 1uM for 4 hours whole cell lysate10 µg (Input).
Lane 2: ab202068 IP in HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202068 in HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.Exposure time: 3 seconds.
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All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/50000 dilution
Lane 1 : Untreated HeLa (human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg
Lane 2 : HeLa (human epithelial cells from cervix adenocarcinoma) treated with staurosporine 1 µM for 4 hours whole cell lysate at 20 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 46 kDa
Observed band size: 35,37,46 kDa why is the actual band size different from the predicted?Blocking/Dilution buffer: 5% NFDM/TBST.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (76)
ab202068 has been referenced in 76 publications.
- Gan RH et al. High expression of Notch2 drives tongue squamous cell carcinoma carcinogenesis. Exp Cell Res 399:112452 (2021). PubMed: 33382997
- Shan G et al. Downregulated exosomal microRNA-148b-3p in cancer associated fibroblasts enhance chemosensitivity of bladder cancer cells by downregulating the Wnt/ß-catenin pathway and upregulating PTEN. Cell Oncol (Dordr) 44:45-59 (2021). PubMed: 33423167
- Gui C et al. Total flavone extract from Ampelopsis megalophylla induces apoptosis in the MCF-7 cell line. Int J Oncol 58:409-418 (2021). PubMed: 33469684
- Li Y et al. miRNA-128 modulates bone neoplasms cells proliferation and migration through the WNT/ß-catenin and EMT signal pathways. J Orthop Surg Res 16:71 (2021). PubMed: 33472642
- Liu Z et al. Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma. Cell Death Dis 12:164 (2021). PubMed: 33558476