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|Sample type||Average %||Range|
|Cell culture media||99||93% - 107%|
|Fetal Bovine Serum||93||85% - 104%|
p53 human SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) in vitro kit (ab171571) is designed for the accurate quantitative measurement of p53 protein in human cell samples.
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.
p53 (TP53 gene) acts as a tumor suppressor in many tumor types and induces growth arrest or apoptosis depending on the physiological circumstances and cell type. p53 is involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. One of the activated genes is an inhibitor of cyclin-dependent kinases. p53 mediated apoptosis induction seems to be by stimulation of BAX and FAS antigen expression, or by repression of Bcl-2 expression. p53 is also implicated in Notch signaling crossover
The p53 protein is found in increased amounts in a wide variety of transformed cells. p53 is mutated or inactivated in about 60% of cancers. Four types of cancers account for 80% of tumors occurring in TP53 germline mutation carriers: breast cancers, soft tissue and bone sarcomas, brain tumors (astrocytomas) and adrenocortical carcinomas.
p53 levels are kept low through a continuous degradation of p53. Mdm2 binds to p53, preventing its action and transports it from the nucleus to the cytosol. Mdm2 also acts as ubiquitin ligase and covalently attaches ubiquitin to p53 and thus marks p53 for degradation by the proteasome. The ubiquitin can be cleaved by USP7 (or HAUSP), thereby protecting it from this proteasome dependent degradation. This is one means by which p53 is stabilized in response to oncogenic insults.
Phosphorylation of the N-terminal end of p53, and conformational changes to p53, disrupt Mdm2-binding leading to p53 accumulation. Acetylation of the C-terminal end of p53 exposes the DNA binding domain of p53, allowing it to activate or repress specific genes.
Deacetylase enzymes, such as Sirt1 and Sirt7, can deacetylate p53, leading to an inhibition of apoptosis.
|Components||1 x 96 tests|
|10X p53 Capture Antibody||1 x 600µl|
|10X p53 Detector Antibody||1 x 600µl|
|10X Wash Buffer PT (ab206977)||1 x 20ml|
|4X Antibody Diluent EB||1 x 6ml|
|50X Cell Extraction Enhancer Solution (ab193971)||1 x 1ml|
|5X Cell Extraction Buffer PTR (ab193970)||1 x 10ml|
|p53 Human Lyophilized Recombinant Protein||2 x 0.05µg|
|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 ab171571 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.|
Background subtracted data from duplicate measurements are plotted. Note the relative lower expression of p53 in the MCF7, HeLa and HL60 cell lines compared to HEK293 and SH-SY5Y.
ELISA (barchart) and western blot (top). Raw OD450 nm data from duplicate measurements for the indicated cells lines is shown (20 µg lysate analyzed). The p53 capture antibody was used to blot the same lysates as analyzed by ELISA (20 µg loaded/lane). The GAPDH blot is included to show the relative loads of each lysate.
ab171571 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"