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|Sample type||Average %||Range|
|Cell culture media||104||98% - 109%|
|Extraction Buffer||107||106% - 109%|
|Goat Serum||93||92% - 93%|
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).
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 (Asp175) Detector Antibody||1 x 700µl|
|10X Blocking Buffer||1 x 6ml|
|10X HRP Label||1 x 1ml|
|10X Wash Buffer||1 x 40ml|
|20X Buffer||1 x 3ml|
|2X Extraction Buffer||1 x 15ml|
|Caspase 3 Microplate||1 unit|
|HeLa-Staurosporine Standard||1 x 400µg|
|HRP Development Solution||1 x 12ml|
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.
|ELISA||Use at an assay dependent concentration.|
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.
ab168541 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"