Human Active Caspase-3 Ser29 ELISA Kit (ab181418)
Key features and details
- One-wash 90 minute protocol
- Sensitivity: 58 pg/ml
- Range: 0.156 ng/ml - 10 ng/ml
- Sample type: Cell culture extracts, Tissue Extracts
- Detection method: Colorimetric
- Assay type: Sandwich (quantitative)
- Reacts with: Human
Overview
-
Product name
Human Active Caspase-3 Ser29 ELISA Kit -
Detection method
Colorimetric -
Precision
Intra-assay Sample n Mean SD CV% Overall 5 2.1% Inter-assay Sample n Mean SD CV% Overall 3 7.1% -
Sample type
Cell culture extracts, Tissue Extracts -
Assay type
Sandwich (quantitative) -
Sensitivity
58 pg/ml -
Range
0.156 ng/ml - 10 ng/ml -
Recovery
106 %
Sample specific recovery Sample type Average % Range Cell culture media 106 100% - 110% -
Assay time
1h 30m -
Assay duration
One step assay -
Species reactivity
Reacts with: Human -
Product overview
Human Active Caspase 3 (Ser29) ELISA kit (ab181418) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of p17 subunit of Active Caspase-3 protein in human cell and tissue extracts. It uses our proprietary SimpleStep ELISA® technology. Quantitate human Active Caspase 3 with 58 pg/mL sensitivity.
SimpleStep ELISA® technology employs capture antibodies conjugated to an affinity tag that is recognized by the monoclonal antibody used to coat our SimpleStep ELISA® plates. This approach to sandwich ELISA allows the formation of the antibody-analyte sandwich complex in a single step, significantly reducing assay time. See the SimpleStep ELISA® protocol summary in the image section for further details. Our SimpleStep ELISA® technology provides several benefits:
-Single-wash protocol reduces assay time to 90 minutes or less
-High sensitivity, specificity and reproducibility from superior antibodies
-Fully validated in biological samples
-96-wells plate breakable into 12 x 8 wells strips
A 384-well SimpleStep ELISA® microplate (ab203359) is available to use as an alternative to the 96-well microplate provided with SimpeStep ELISA® kits. -
Notes
Caspase-3 is a cysteine protease involved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis Caspase-3 proteolytically cleaves poly (ADP-ribose) polymerase (PARP) at Asp216-Gly217 bond. Caspase-3 cleaves and activates sterol regulatory element binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. Caspase-3 cleaves and activates caspase-6, -7 and -9. Caspase-3 is involved in the cleavage of huntingtin. Caspase-3 is a cytoplasmic protein highly expressed in lung, spleen, heart, liver and kidney. Moderate levels of Caspase-3 are in brain and skeletal muscle, and low levels in testis. Also Caspase-3 is found in many cell lines, highest expression in cells of the immune system. 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 active subunits. Thus the pro-form and the active form are useful biomarkers of apoptosis. Active Caspase-3 is a heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 17 kDa (p17) and a 12 kDa (p12) subunit. 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. Caspase-3 is 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.
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses. -
Platform
Microplate
Properties
-
Storage instructions
Store at +4°C. Please refer to protocols. -
Components 1 x 96 tests 10X Human Active Caspase-3 (Ser29) Capture Antibody 1 x 600µl 10X Human Active Caspase-3 (Ser29) 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 4BI 1 x 6ml Human Active Caspase-3 Lyophilized Recombinant Protein 2 vials Plate Seals 1 unit Sample Diluent NS (ab193972) 1 x 12ml SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit Stop Solution 1 x 12ml TMB Development Solution 1 x 12ml -
Research areas
-
Cellular localization
Cytoplasmic -
Alternative names
- Apopain
- CASP 3
- CASP3
see all -
Database links
- Entrez Gene: 836 Human
- Omim: 600636 Human
- SwissProt: P42574 Human
- Unigene: 141125 Human
Images
-
Example of Active Caspase-3 standard curve
Example of Active Caspase-3 standard curve for measurement of extracts prepared from cell pellets and tissue homogenates. Background-subtracted data values (mean +/- SD, n=2) are graphed.
-
Other - Human Active Caspase-3 Ser29 ELISA Kit (ab181418)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.
-
Example of Active Caspase-3 standard curveExample of Active Caspase-3 standard curve for measurement of lysates prepared from cells in media (RPMI1640 supplemented with 10% Fetal Bovine Serum) by direct in well lysis. Background-subtracted data values (mean +/- SD, n=2) are graphed.
-
Titration of Jurkat extractTitration of Jurkat Cell Extract prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or vehicle (DMSO) within the working range of the assay. Raw data values (mean +/- SD, n=2) are graphed. Dotted line represents Blank control.
-
Titration of HeLa extractTitration of HeLa Cell Extract prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or vehicle (DMSO) within the working range of the assay. Raw data values (mean +/- SD, n=2) are graphed. Dotted line represents Blank control.
-
Example of staurosporine IC50 determinationExample of Staurosporine IC50 determination. Jurkat Cell Lysates corresponding to 2x106 cells/mL or 1x106 cells/mL were prepared by direct in-well lysis (without media removal) from cells grown in RPMI1640 media supplemented with 10% FBS and treated for 4 hours with variable doses of Staurosporine in a 96-well plate. Background-subtracted data values (mean +/SD, n=3) are graphed. IC50 determined from background-subtracted data were 1.1 µM and 0.9 µM using, respectively, lysates of 2x106 cells/mL and 1x106 cells/mL.
-
Comparison of active caspase-3 concentration in Jurkat cell extractsComparison of active caspase 3 concentration in Jurkat Cell Extracts prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or drug’s vehicle (DMSO) using Active Caspase 3 (Ser29) Human SimpleStep™ ELISA Kit. Background-subtracted data values (mean +/- SD, n=2) of several extract concentrations analyzed (as indicated) are graphed. Note that the Active Caspase 3 is detectable only in cells undergoing apoptosis induced by Staurosporine treatment. This result correlates well with Western blot analysis.
-
Quantification of active caspase-3 concentration in Jurkat cell extractsQuantification of Active Caspase-3 concentration in Jurkat Cell Extracts prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or drug’s vehicle (DMSO) using Active Caspase 3 (Ser29) Human SimpleStep™ ELISA Kit. The concentrations of Active Caspase 3 were interpolated from data values shown in Figure 6 using Active Caspase 3 standard curve, corrected for sample dilution, and graphed in ng of Active Caspase 3 per mg of extract. Note that the Active Caspase 3 is detectable only in cells undergoing apoptosis induced by Staurosporine treatment. This result correlates well with Western blot analysis.
-
Figure 8Demonstration of the capture and detector antibodies specificities. Active Caspase 3 protein (ab52314, lane 1, 8 ng/lane) and 20 µg/lane of cell extracts prepared from 4 hours vehicle-treated (lane 2) and 1 µM Staurosporine-treated (lane 3) Jurkat cells were analyzed by Western blotting using the Active Caspase 3 (Ser29) Capture Antibody (A), or the Active Caspase 3 (Ser29) Detector Antibody (B). Note that the Active Caspase 3 (Ser29) Capture Antibody used in this kit specifically detects only the p17 subunit of Active Caspase 3 but not the pro-caspase 3 in Jurkat Cell Extracts.
