Human Cleaved Caspase-3 (Asp175) ELISA Kit (ab220655)
- Datasheet
- References
- Protocols
Overview
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Product name
Human Cleaved Caspase-3 (Asp175) ELISA Kit
See all Cleaved Caspase-3 kits -
Detection method
Colorimetric -
Precision
Intra-assay Sample n Mean SD CV% Cell extract 3 4.2% Inter-assay Sample n Mean SD CV% Cell extract 5 5.2% -
Sample type
Cell culture extracts, Tissue Extracts -
Assay type
Sandwich (quantitative) -
Sensitivity
2.1 pg/ml -
Range
15.63 pg/ml - 1000 pg/ml -
Recovery
Sample specific recovery Sample type Average % Range Cell culture extracts 108 105% - 115% -
Assay time
1h 30m -
Assay duration
One step assay -
Species reactivity
Reacts with: Human -
Product overview
Cleaved Caspase-3 (Asp175) in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Active Caspase-3 (Asp175) protein in human cell and tissue extract samples.
The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
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Notes
Caspase-3 is a cytoplasmic cysteine protease involved in the activation cascade of caspases responsible for execution of apoptosis. At the onset of apoptosis caspase-3 cleaves and activates caspase-6, -7 and -9. It also cleaves poly (ADP-ribose) polymerase (PARP). Caspase-3 cleaves and activates sterol regulatory element binding proteins (SREBPs). Caspase-3 is involved in the cleavage of huntingtin. Caspase-3 is expressed in an inactive pro-form (pro caspase-3). In apoptosis, the pro caspase-3 is activated by proteolytic cleavages at Asp28-Ser29 and Asp175-Ser176 bonds catalyzed by granzyme B, caspase-6, caspase-8, caspase-9 and caspase-10, generating two subunits p17 and p12 that are assembled into heterotetrameric active enzyme. Additional processing of the propeptides is likely due to the autocatalytic activity of the activated protease. The pro-form and the active form are useful biomarkers of apoptosis.
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Tested applications
Suitable for: Sandwich ELISAmore details -
Platform
Microplate (12 x 8 well strips)
Properties
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Storage instructions
Store at +4°C. Please refer to protocols. -
Components 1 x 96 tests 10X Human Cleaved Caspase-3 (Asp175) Capture Antibody 1 x 600µl 10X Human Cleaved Caspase-3 (Asp175) Detector Antibody 1 x 600µl 10X Wash Buffer PT (ab206977) 1 x 20ml 50X Cell Extraction Enhancer Solution (ab193971) 1 x 1ml 5X Cell Extraction Buffer PTR (ab193970) 1 x 10ml Antibody Diluent CPI - HAMA Blocker (ab193969) 1 x 6ml Human Cleaved Caspase-3 (Asp175) Lyophilized Recombinant Protein 2 vials Plate Seals 1 unit Sample Diluent NS (ab193972) 1 x 12ml SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 x 96 tests Stop Solution 1 x 12ml TMB Development Solution 1 x 12ml -
Research areas
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Function
Involved 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. Triggers cell adhesion in sympathetic neurons through RET cleavage. -
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. -
Sequence similarities
Belongs to the peptidase C14A family. -
Post-translational
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. -
Cellular localization
Cytoplasm. - Information by UniProt
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Alternative names
- active caspase 3
- Apopain
- CASP 3
see all -
Database links
- Entrez Gene: 836 Human
- Omim: 600636 Human
- SwissProt: P42574 Human
- Unigene: 141125 Human
Associated products
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Alternative Versions
Applications
Our Abpromise guarantee covers the use of ab220655 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 |
|---|---|---|
| Sandwich ELISA | Use at an assay dependent concentration. |
Images
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Other - Human Cleaved Caspase-3 (Asp175) ELISA Kit (ab220655)SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.
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Example of human Active Caspase-3 (Asp175) standard curve in 1X Cell Extraction Buffer PTR.Background-subtracted data values (mean +/- SD) are graphed.
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Interpolated concentrations of native Active Caspase-3 (Asp175) in staurosporine treated Jurkat cell extract samples collected at different time points.Jurkat cells were cultured in the presence of 1 µM staurosporine and collected at time points of 2 hours, 4 hours and 6 hours based on a 10 μg/mL extract load. The concentrations of Active Caspase-3 (Asp175) were measured in duplicate and interpolated from the Active Caspase-3 (Asp175) standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Active Caspase-3 (Asp175) concentration was determined to be 206.0 pg/mL in staurosporine treated Jurkat (2 hour), 472.9 pg/mL in staurosporine treated Jurkat (4 hour) and 376.7 pg/mL in staurosporine treated Jurkat (6 hour) cell extracts.
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Comparison of staurosporine treated Jurkat cell extracts collected at different time points during treatment.Jurkat cells were cultured in the presence of 1 µg/mL staurosporine and collected at time points of 2 hours, 4 hours and 6 hours, or in the presence of staurosporine solvent (mock) collected at 4 hours. The concentration of Active Caspase-3 (Asp175) was measured in three different dilutions of the cell extract samples in duplicate and interpolated from the Active Caspase-3 (Asp175) standard curve. The interpolated dilution factor corrected values are plotted in ng of Active Caspase-3 (Asp175) per mg of extract (mean +/- SD, n=3). The mean Active Caspase-3 (Asp175) concentration was determined to be not detectable in Jurkat (mock treated), 21.16 ng/mg in Jurkat (2 hour), 49.00 ng/mg in Jurkat (4 hour) and 38.79 ng/mg in Jurkat (6 hour) cell extracts.
Protocols
References
ab220655 has not yet been referenced specifically in any publications.
