Overview

  • Product name
    Human Active Caspase 3 ELISA Kit (Asp175)
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    Overall 3 3.4%
    Inter-assay
    Sample n Mean SD CV%
    Overall 3 6.7%
  • Sample type
    Cell culture extracts, Adherent cells, Suspension cells, Tissue Extracts, Tissue Homogenate
  • Assay type
    Sandwich (quantitative)
  • Sensitivity
    2 µg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture media 104 98% - 109%
    Extraction Buffer 107 106% - 109%
    Goat Serum 93 92% - 93%

  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Human
  • Product overview

    Abcam’s Active Caspase 3 (Asp175) Human in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the accurate quantitative measurement of p17 subunit of active caspase 3 protein in human cells and tissue homogenates

    The assay employs an antibody specific to caspase 3 protein coated onto well plate strips. Standards and samples are pipetted into the wells and caspase 3 present in the sample is bound to the wells by the immobilized antibody. The wells are washed and an anti-active caspase 3 (Asp175) detector is added. After washing away unbound detector antibody, HRP-conjugated label specific for the detector antibody is pipetted into the wells. The wells are again washed, a TMB substrate solution is added to the wells and blue color develops in proportion to the amount of p17 subunit of caspase 3. The developing blue color is measured at 600 nm. Optionally the reaction can be stopped by adding hydrochloric acid which changes the color from blue to yellow and the intensity can be measured at 450 nm.

    Since the anti-active caspase 3 (Asp175) detector antibody is specific to the extreme C-terminus of fragment(s) generated by a cleavage between Asp175 and Ser176 of the pro-caspase 3, the assay is specific for the p17 subunit of active caspase 3 (AA 29-175). It thus measures a well recognized biomarker of apoptosis. The assay may also measure partially cleaved forms of caspase 3 (AA 1-175 and AA 10-175). The assay does not detect the pro-form of caspase 3 (full-length caspase 3, AA 1-277), partially cleaved forms of caspase 3 (AA 10-277 and AA 28-277), or p12 subunit of caspase 3 (AA 176-277).

     

  • 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.

     

  • Tested applications
    Suitable for: ELISAmore details
  • Platform
    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab168541 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
ELISA Use at an assay dependent concentration.

Images

  • HeLa-Staurosporine standard curve. The HeLa Staurosporine standard was prepared using HeLa cells treated for 4 hours with 1 µM staurosporine (ab120056). Background-subtracted data values (mean +/- SD) are graphed.

  • Lysates corresponding to 0.33x106 HeLa cells/mL or 0.50x106 Jurkat cells/mL were prepared by direct in-well lysis (without media removal) from cells treated for 4 hours with variable doses of staurosporine in a 96-well plate. 100 µL of the lysates were analyzed in triplicates. Background-subtracted signals are shown in left panels. Relative active caspase 3 concentrations interpolated from standard curves and expressed as percent of cells treated with the highest dose of staurosporine are shown in right panels. IC50 were 0.9 µM for HeLa cells and 0.4 µM for Jurkat cells.
  • Demonstration of the assay specificity by induction of active caspase 3 by staurosporine treatment. Cells were treated for 4 hours with 1 µM staurosporine or drug’s vehicle (DMSO) and 500 µg/mL of HeLa cell extracts and 125 µg/mL Jurkat cell extracts prepared from cell pellets were analyzed using this kit. Relative active caspase 3 levels were interpolated from HeLa-Staurosporine standard curve and expressed as percent of staurosporine-treated HeLa cell. Note that no active caspase 3 (p17 subunit) was detected in vehicle-treated HeLa and Jurkat cells.
  • Cell extracts were prepared from 4 hours vehicle-treated (lanes 1 and 3) and 1 µM staurosporine-treated (lanes 2 and 4) Jurkat cells. Extracts were incubated with the Caspase 3 Microplate, captured proteins were extracted and analyzed by Western blotting using a pro+p17 caspase 3 antibody ab32351 (Lanes 3 and 4). 25% of the amounts of the extracts used for immunocapture were also analyzed directly by the Western blotting (lanes 1 and 2). Note that the Caspase 3 Microplate captures both the pro- and p17 subunit of caspase 3.

Protocols

References

ab168541 has not yet been referenced specifically in any publications.

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