Recombinant protein within Human Caspase-3 aa 9-175 / 277.
Species Reactivity
Human, Mouse, Rat
Validated Applications
WB, IHC-P, IP
Target Molecular Weight
Predicted band size: 32/17 kDa
Positive Control
HeLa cell lysate, HeLa treated with 1μM staurosporine for 4 hours cell lysate, HeLa treated with 3μM staurosporine for 4 hours cell lysate, C6 cell lysate, C6 treated with 1μM staurosporine for 4 hours cell lysate, C6 treated with 3μM staurosporine for 4 hours cell lysate, U-87 MG cell lysate, MDA-MB-231 cell lysate, NIH/3T3 cell lysate, Jurkat cell lysate, Jurkat treated with 1μM staurosporine for 3 hours cell lysate, human lung carcinoma tissue, human spleen tissue, human liver tissue, human kidney tissue.
Conjugation
unconjugated
Clone Number
SU38-04
Product Features
Form
Liquid
Concentration
1mg/ml
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage Buffer
1*PBS (pH7.4).
Isotype
IgG
Purification Method
Protein A affinity purified.
Application Dilution
WB
1:1,000-1:2,000
IHC-P
1:50-1:1,000
IP
Use at an assay dependent concentration.
Target
Function
Caspase-3 is a caspase protein that interacts with caspase-8 and caspase-9. It is encoded by the CASP3 gene. CASP3 orthologs have been identified in numerous mammals for which complete genome data are available. Unique orthologs are also present in birds, lizards, lissamphibians, and teleosts. Caspase-3 shares many of the typical characteristics common to all currently-known caspases. For example, its active site contains a cysteine residue (Cys-163) and histidine residue (His-121) that stabilize the peptide bond cleavage of a protein sequence to the carboxy-terminal side of an aspartic acid when it is part of a particular 4-amino acid sequence. This specificity allows caspases to be incredibly selective, with a 20,000-fold preference for aspartic acid over glutamic acid. A key feature of caspases in the cell is that they are present as zymogens, termed procaspases, which are inactive until a biochemical change causes their activation. Each procaspase has an N-terminal large subunit of about 20 kDa followed by a smaller subunit of about 10 kDa, called p20 and p10, respectively.
Background References
1. Dong L et al. Echinacoside Induces Apoptosis in Human SW480 Colorectal Cancer Cells by Induction of Oxidative DNA Damages. Int J Mol Sci 16:14655-68 (2015).
2. Nilsonne G et al. Phenotype-dependent apoptosis signalling in mesothelioma cells after selenite exposure. J Exp Clin Cancer Res 28:92 (2009).
Sequence Similarity
Belongs to the peptidase C14A family.
Tissue Specificity
Highly 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.
Post-translational Modification
Cleavage 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.
Western blot analysis of Cleaved+pro Caspase-3 on different lysates with Rabbit anti-Cleaved+pro Caspase-3 antibody (HA750159) at 1/1,000 dilution.
Lane 1: HeLa cell lysate Lane 2: HeLa treated with 1μM staurosporine for 4 hours cell lysate Lane 3: HeLa treated with 3μM staurosporine for 4 hours cell lysate Lane 4: C6 cell lysate Lane 5: C6 treated with 1μM staurosporine for 4 hours cell lysate Lane 6: C6 treated with 3μM staurosporine for 4 hours cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 32/17 kDa Observed band size: 32/17 kDa
Exposure time: 3 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750159) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
☑ Knockout (KO)
Western blot analysis of Caspase-3 with anti-Caspase-3 antibody (HA750159) at 1:2,000 dilution. Lane 1: Wild-type Hela whole cell lysate (10 µg). Lane 2: Caspase-3 knockout Hela whole cell lysate (10 µg).
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (HA750159, 1/2,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
☑ Cell treatment (CT)
Western blot analysis of Cleaved+pro Caspase-3 on different lysates with Rabbit anti-Cleaved+pro Caspase-3 antibody (HA750159) at 1/5,000 dilution.
Lane 1: U-87 MG cell lysate Lane 2: MDA-MB-231 cell lysate Lane 3: NIH/3T3 cell lysate Lane 4: Jurkat cell lysate Lane 5: Jurkat treated with 1μM staurosporine for 3 hours cell lysate
Lysates/proteins at 15 µg/Lane.
Predicted band size: 32/17 kDa Observed band size: 32/17 kDa
Exposure time: 1 minute 2 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750159) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded human lung cancer tissue with Rabbit anti-Cleaved+pro Caspase-3 antibody (HA750159) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750159) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-Cleaved+pro Caspase-3 antibody (HA750159) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750159) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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