The caspase family of cysteine proteases plays a key role in apoptosis and inflammation. Caspase-3 is a key protease that is activated during the early stages of apoptosis and, like other members of the caspase family, is synthesized as an inactive pro-enzyme that is processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by another protease. The processed forms of caspases consist of large (17-22 kDa) and small (10-12 kDa) subunits which associate to form an active enzyme. Active caspase-3, a marker for cells undergoing apoptosis, consists of a heterodimer of 17 and 12 kDa subunits which is derived from the 32 kDa pro-enzyme. Active caspase-3 proteolytically cleaves and activates other caspases, as well as relevant targets in the cytoplasm, e.g., D4-GDI and Bcl-2, and in the nucleus (e.g. PARP). This antibody has been reported to specifically recognize the active form of caspase-3 in human and mouse cells. It has not been reported to recognize the pro-enzyme form of caspase-3. Flow cytometric analysis of apoptotic and non-apoptotic populations for active caspase-3. Jurkat cells (Human T-cell leukemia, ATCC TIB-152) were left untreated (left panel) or treated with 4 μM of camptothecin for 4 hr to induce apoptosis (right panel). Cells were washed once in PBS, then fixed and permeabilized using the BD Cytofix/Cytoperm™ Kit (Cat. No. 554714) for 20 min at room temperature (RT), pelleted and washed with BD Perm/Wash™ buffer (component of Cat. No. 554714). Cells were subsequently stained with the biotin rabbit anti- active caspase-3 antibody (clone C92-605). Cells were then washed and resuspended in BD Perm/Wash™ buffer containing FITC-Avidin (5 ng/1x10e6 cells) [Cat. No. 554057] and incubated for 30 min at RT in the dark. Cells were then washed and resuspended in BD Perm/Wash™ buffer before analyzing by flow cytometry. The results show that untreated cells were primarily negative for active caspase-3 (left panel, M1), whereas more than half of the treated cells were positive for active caspase-3 staining (right panel, M2).
CASP3
Reactivity: Human
WB, IHC, IF
Host: Rabbit
Polyclonal
unconjugated
Application Notes
Optimal working dilution should be determined by the investigator.
Sample Volume
20 μL
Restrictions
For Research Use only
Buffer
Aqueous buffered solution containing BSA and ≤0.09 % sodium azide.
Preservative
Sodium azide
Precaution of Use
This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice
The antibody was conjugated with biotin under optimum conditions, and unreacted biotin was removed.
Storage
4 °C
Storage Comment
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Dai, Krantz: "Interferon gamma induces upregulation and activation of caspases 1, 3, and 8 to produce apoptosis in human erythroid progenitor cells." in: Blood, Vol. 93, Issue 10, pp. 3309-16, (1999) (PubMed).
Fujita, Tsuruo: "Involvement of Bcl-2 cleavage in the acceleration of VP-16-induced U937 cell apoptosis." in: Biochemical and biophysical research communications, Vol. 246, Issue 2, pp. 484-8, (1998) (PubMed).
Thornberry, Lazebnik: "Caspases: enemies within." in: Science (New York, N.Y.), Vol. 281, Issue 5381, pp. 1312-6, (1998) (PubMed).