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|Sample type||Average %||Range|
|Cell culture media||106||100% - 110%|
Active Caspase 3 (Ser29) in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of p17 subunit of Active Caspase 3 (Ser29) protein in Human cell and tissue extracts.
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.
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.
|Components||1 x 96 tests|
|10X Active Caspase 3 (Ser29) Capture Antibody||1 x 600µl|
|10X 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||1 x 10ml|
|Active Caspase 3 Human Lyophilized Recombinant Protein||2 vials|
|Antibody Diluent 4BI||1 x 6ml|
|Plate Seals||1 unit|
|Sample Diluent NS||1 x 12ml|
|SimpleStep Pre-Coated 96-Well Microplate (ab206978)||1 unit|
|Stop Solution||1 x 12ml|
|TMB Development Solution||1 x 12ml|
Our Abpromise guarantee covers the use of ab181418 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Sandwich ELISA||Use at an assay dependent concentration.|
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.
Example 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 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 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 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 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 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.
Demonstration 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.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"