Product nameAnti-Caspase-3 antibody
See all Caspase-3 primary antibodies
DescriptionRabbit polyclonal to Caspase-3
Stimulation is required to allow detection of the 17kDa cleaved form of the protein. Please see images below for recommended treatment conditions and positive controls.
ab13847 recognizes a cleaved form of Caspase 3 (~17 kDa) after apoptosis has been induced in wildtype cells and not Caspase 3 knockout cells.
Some customers have used this antibody successfully in IHC-P however our latest tests were unsuccessful and therefore we can no longer guarantee this application. We would recommend ab32351 and ab184787 as an alternative product for this application.
Tested applicationsSuitable for: ICC/IF, IHC-Fr, WB, Flow Cyt, ICC, IHC - Wholemountmore details
Species reactivityReacts with: Mouse, Rat, Human, Pig, Xenopus laevis, Drosophila melanogaster, Indian muntjac, Zebrafish, Rhesus monkey, Common marmoset, Schmidtea mediterranea, Salvelinus alpinus
Predicted to work with: Dog, Chinese hamster
- WB: HAP1 cell lysate; human caspase-3 recombinant protein. Flow Cyt: Jurkat cells. IHC-Fr: Tumour tissue; Rat brain tissue with a kainite lesion. ICC/IF: HeLa cells.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
Storage bufferpH: 7.40
Preservative: 0.02% Sodium azide
Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
Concentration information loading...
PurityImmunogen affinity purified
Our Abpromise guarantee covers the use of ab13847 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 5 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
|WB||1/500. Detects a band of approximately 17, 34 kDa (predicted molecular weight: 17, 34 kDa).|
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
|ICC||Use at an assay dependent concentration.|
|IHC - Wholemount||1/500.|
FunctionInvolved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis it proteolytically cleaves poly(ADP-ribose) polymerase (PARP) at a '216-Asp-
-Gly-217' bond. Cleaves and activates sterol regulatory element binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. Cleaves and activates caspase-6, -7 and -9. Involved in the cleavage of huntingtin.
Tissue specificityHighly expressed in lung, spleen, heart, liver and kidney. Moderate levels in brain and skeletal muscle, and low in testis. Also found in many cell lines, highest expression in cells of the immune system.
Sequence similaritiesBelongs to the peptidase C14A family.
modificationsCleavage by granzyme B, caspase-6, caspase-8 and caspase-10 generates the two active subunits. Additional processing of the propeptides is likely due to the autocatalytic activity of the activated protease. Active heterodimers between the small subunit of caspase-7 protease and the large subunit of caspase-3 also occur and vice versa.
S-nitrosylated on its catalytic site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway, associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active site thiol.
- Information by UniProt
- A830040C14Rik antibody
- Apopain antibody
- CASP 3 antibody
Lane 1: Wild-type HAP1 cell lysate + Staurosporine (ab146588) (1μM for 4h)
Lane 2: Wild-type HAP1 cell lysate
Lane 3: Caspase-3 knockout HAP1 cell lysate + Staurosporine (ab146588) (1μM for 4h)
Lane 4: Caspase-3 knockout HAP1 cell lysate
Lanes 1 - 4: Merged signal (red and green). Green - ab13847 observed at 17 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab13847 was shown to recognise Caspase 3 when Caspase 3 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase 3 knockout samples (±staurosporine treatment) were subjected to SDS-PAGE. ab13847 and ab8245 (loading control to GAPDH) were diluted to 1/500 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
5 μm frozen sections of tumor tissue were fixed with 100% ice cold methanol for 10 minutes, then blocked in 5% normal goat serum in PBS (pH 7.4) for 1h. Sections were incubated with ab13847 (1:500) at 4°C overnight and for 1h with secondary antibodies at room temperature.
All lanes : Anti-Caspase-3 antibody (ab13847) at 1 µg/ml
Lane 1 : Human Caspase 3 (active) Recombinant Protein
Lane 2 : Human Pro Caspase 3 (inactive) Recombinant Protein
Lysates/proteins at 0.1 µg per lane.
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 17, 34 kDa
Observed band size: 17,32 kDa why is the actual band size different from the predicted?
Exposure time: 8 minutes
Caspase 3 exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce large (17kDa) and small (12kDa) subunits. These subunits dimerize to form the active enzyme. ab13847 specifically detects the large active subunit (17kDa) and the inactive pro Caspase 3 (32 kDa).
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab13847 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.
Secondary antibody - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody
ab13847 staining active caspase 3 in Human Jurkat cells by Flow Cytometry. Cells were prepared in a phosphate buffered solution containing 0.1% sodium azide with FBS fixed with paraformaldehyde and permeabilized with Triton X-100 and NP40. The sample was incubated with the primary antibody (1/100 in wash buffer) for 24 hours at 4°C. A FITC-conjugated Goat anti-rabbit Ig (1/100) was used as the secondary antibody.
Gating Strategy: Isolate cell population from plot of SSC-A / FSA-A
ab13847 was used in IHC of frozen sections from a rat brain with a kainite lesion. The non lesionned contralateral site serves as a negative control. The sections were fixed with paraformaldehyde. The tissue was perfused with 4% PFA and embedded in OCT compound and cut on the cryostat. The primary antibody was incubated for 12 hours at a dilution of 1/300. A biotin labelled secondary antibody was used at a dilution of 1/300.
HeLa cells were fixed for 10 minutes at room temperature in 3.7% PFA and permeabilised in 0.1% Triton X-100/PBS then incubated with ab13847 (5µg/ml) for 1 hour at room temperature. The top panel shows control cells treated with DMSO. The bottom panel shows HeLa cells treated with 1 mM staurosporine (ab146588) for 4 hours to induce caspase-3 activation. ab13847 staining is shown in green and counterstaining with DAPI is shown in blue. 100x magnification.
The image shows the staining with ab13847 is very faint in the untreated control cultures, but very bright after activation of capsase-3 by treatment with the staurosporine . (N.B. in these cultures the nuclei are apoptotic).
This product has been referenced in:
- Xi X et al. MicroRNA-204-3p represses colon cancer cells proliferation, migration, and invasion by targeting HMGA2. J Cell Physiol 235:1330-1338 (2020). Read more (PubMed: 31286521) »
- Yang Y et al. Skullcapflavone I suppresses proliferation of human lung cancer cells via down-regulating microRNA-21. Exp Mol Pathol 110:104285 (2019). Read more (PubMed: 31301304) »