Overview

  • Product name
    Human Active Caspase-3 (Asp175) ELISA Kit
    See all active 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

    Active 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.


     

  • 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.

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate (12 x 8 well strips)

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Human Active Caspase-3 (Asp175) Capture Antibody 1 x 600µl
    10X Human Active 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 1 x 6ml
    Human Active Caspase-3 (Asp175) Lyophilized Recombinant Protein 2 vials
    Plate Seals 1 unit
    Sample Diluent NS 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
  • 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
    modifications
    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.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Alternative names
    • Apopain
    • CASP 3
    • CASP-3
    • CASP3
    • CASP3_HUMAN
    • Caspase 3
    • Caspase-3 subunit p12
    • cleaved caspase 3
    • CPP 32
    • CPP-32
    • CPP32B
    • Cysteine protease CPP32
    • PARP cleavage protease
    • Protein Yama
    • SCA-1
    • SCA1
    • SREBP cleavage activity 1
    • Yama
    see all
  • Database links

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

  • Background-subtracted data values (mean +/- SD) are graphed.

  • 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.

  • 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.

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