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|Sample type||Average %||Range|
|Cell culture media||59||43% - 73%|
|Fetal Bovine Serum||65||52% - 85%|
|Bovine Serum Albumin||75||57% - 94%|
Abcam’s cleaved PARP1 in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of cleaved PARP1 protein in human cell 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.
PARP1 is a 113 kDa nuclear DNA-repair enzyme that transfers ADP-ribose units from NAD+ to variety of nuclear proteins including topoisomerases, histones and PARP1 itself. Via poly ADP ribosylation, PARP1 is responsible for regulation of cellular homeostasis including cellular repair, transcription and replication of DNA, cytoskeletal organization and protein degradation. In response to DNA damage, PARP1 activity is increased upon binding to DNA strand nicks and breaks. Excessive DNA damage leads to generation of large branched ADP-ribose polymers and activation of a unique cell death program.
During apoptosis, PARP1 is cleaved by activated caspase-3 between Asp214 and Gly215, resulting in the formation of an N-terminal 24 kDa fragment containing most of the DNA binding domain and a C-terminal 89 kDa fragment containing the catalytic domain. The proteolysis of PARP1 through this cleavage renders the enzyme inactive and this further facilitates apoptotic cell death. Thus the presence of 89 kDa PARP1 fragment is considered to be a very reliable biomarker of apoptosis.
|Components||1 x 96 tests|
|10X Cleaved PARP1 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 5B||1 x 6ml|
|Cleaved PARP1 Capture Antibody||1 x 2 vials|
|HeLa (Staurosporin-treated) Lyophilized Extract||2 x 250µg|
|HeLa (untreated) Lyophilized Extract||2 x 250µg|
|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 ab174441 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.|
Untreated and staurosporine (ab120056) treated HeLa and Jurkat lysates were prepared in 1X Cell Extraction Buffer PTR and tested using the Cleaved PARP1 SimpleStepELISA. Raw OD 450 nm values are shown for 500 µg/mL lysate loads.
HeLa cells were treated with a dose titration of Staurosporine for 4 hours in complete media. Cells were cultured and treated in a 96-well cell culture microtiter plate. Lysates were prepared by direct in-well lysis without media removal: 2X Cell Extraction Buffer PTR was added to an equal volume of media and then resulting lysate was used directly in the Cleaved PARP1 SimpleStepTM ELISA assay. Raw values for triplicate measurements are plotted. The calculated IC50 is 0.77 µM.
20 µg of HeLa extracts that were untreated or treated for 4 hours with 1 µM Staurosporine were analyzed by western blot. The GAPDH blot is included to show the relative loads of each lysate. In the HeLa cell line, Staurosporine treatment is required to detect cleaved PARP1 protein, as observed in the SimpleStep ELISA (Figure 2).
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"