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Phospho-p53 (Ser15) & Total p53 Cellular Assay Kits   Phospho- and total p53 assays for monitoring cellular response to DNA damage These cell-based assays are designed to monitor the modulation of total and phosphorylated p53 (Serine 15). Buffer compatibility means you can use both assays on the same lysate (in parallel) to normalize your results. Due to its tumor suppressor action in many tumor types, P53 has been described as "the guardian of the genome" because of its role in safeguarding genome integrity. P53 has many anticancer function mechanisms, thereby inducing apoptosis, senescence, cell cycle regulation, growth arrest, genomic stability, angiogenesis inhibition, and metabolism changes by regulating the expression of various target genes.   Features Ready-to-use kit: all the necessary reagents included High sensitivity: validated on endogenous levels of phospho- and total P53 in MCF7 or HEK cells Useful complements to JNK and P38 for cell signaling studies, H2AX and cleaved PPARP for apoptotic effects More convenient and faster than ELISA or WB   Applications Oncology Studies of genome stability, cell cycle and genotoxic effects Quantification of cells undergoing apoptosis Efficiency monitoring of anti-cancer treatment      Assay Principle HTRF® - the homogeneous cell-based sandwich immunoassay  Cisbio Bioassay’s phospho- and total p53 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-p53 antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with p53, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.   The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-p53 can be quantitatively detected using the HTRF phospho-p53 (Ser15) and total p53 cellular assay kit reagents and most TR-FRET multimode plate readers.     A simpler, more flexible assay protocol - adapted to your applications Two-plate assay protocol For added flexibility, the assay can be run under a two-plate assay protocol, where cells are first plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.     Simplified pathway Function and regulation of p53 P53 becomes activated in response to myriad stressors, including but not limited to DNA damage (induced by either UV, IR, or chemical agents such as hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion, and deregulated oncogene expression. In response to such stresses, p53 undergoes extensive posttranslational modifications, including phosphorylation and acetylation.    Phosphorylation of p53 mostly occurs in the N-terminal activation domain, where various phosphorylation sites have been described. Among them Ser15 leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2, which inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation.   P53 can be phosphorylated at Ser15 by ATM, ATR directly or through Chk1 and Chk2, DNA-PK and by P38. Phosphorylation in this site impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage.         Product Performance 1. HTRF assay compared to Western Blot using phospho & total P53 cellular assays on human MCF-7 cells Human MCF-7 cells were plated at 106 cells/ well in a 6 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 5 min under UV (2.1J/cm²), the cells were lysed with 200 µL of lysis buffer for 30min at RT under gentle shaking. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-p53 (Ser15) detection reagents. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.   For both HTRF assays (phospho- and total), just 5 000 cells were sufficient for minimal signal detection, while 20 000 cells were needed for a Western Blot signal detection, showing that the two HTRF® p-53 assays are at least 4-fold more sensitive than the Western Blot.       2. Validation on human breast cancer MCF-7 cells treated with Neocarcinostatin Human MCF-7 cells were plated at 100,000 cells/ well in a 96 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 45 min with increasing concentrations of Neocarcinostatin, the medium was removed and the cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-p53 (Ser15) or total p53 detection reagents were added. The HTRF signal was recorded after an overnight incubation.          3. Validation on human breast cancer MCF-7 cells treated with Etoposide, another apoptosis inductor Human MCF-7 cells were plated at 200,000 cells/ well in a 96 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 3 h with increasing concentrations of Etoposide, the medium was then removed and the cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-p53 (Ser15) or total p53 detection reagents were added. The HTRF signal was recorded after an overnight incubation.          

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