Overview

  • Product name
    Human Pro Caspase 3 ELISA Kit
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    Jurkat 5 5.2%
    Inter-assay
    Sample n Mean SD CV%
    Jurkat 3 4.9%
  • Sample type
    Cell culture extracts, Tissue Extracts, Cell Lysate
  • Assay type
    Sandwich (quantitative)
  • Sensitivity
    3 pg/ml
  • Range
    0.156 ng/ml - 10 ng/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture media 73.3 70.7% - 76.1%

  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Human
  • Product overview

    Pro Caspase 3 in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Pro Caspase 3 protein in human cell and tissue extract samples. (UniprotID: P42574)


    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.


    General sensitivity:


    3 pg/mL for measurement of lysates prepared by direct in well lysis.
    11 pg/mL for measurement of extracts prepared from cell pellets and tissue homogenates.

  • 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 bondscatalyzed 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: Sandwich ELISAmore details
  • Platform
    Microplate (12 x 8 well strips)

Properties

Applications

Our Abpromise guarantee covers the use of ab192146 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.

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

  • Background-subtracted data values (mean +/- SD, n = 2) are graphed.

  • Jurkat cell lysates corresponding to 2x106 cells/mL or 1x106 cells/mL were prepared as previously described. The concentrations of Pro Caspase 3 were interpolated from data values shown in the previous figure using Pro Caspase 3 standard curve and graphed in ng of Pro Caspase 3 per 106 cells. Staurosporine IC50 determined from the interpolated data values were 0.70 µM and 0.56 µM using, respectively, lysates of 2x106 cells/mL and 1x106 cells/mL.

  • 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. Staurosporine IC50 determined from background-subtracted data values were 0.75 µM and 0.57 µM using, respectively, lysates of 2x106 cells/mL and 1x106 cells/mL.

Protocols

References

ab192146 has not yet been referenced specifically in any publications.

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