Phospho-p53 (S9) Sandwich ELISA Kit

Transcription

1 Phospho-p53 (S9) Sandwich ELISA Kit Catalog No. KE2037 Detection and Quantification of Phospho-p53 (S9) Concentrations in Cell Lysates. Research Purposes Only. Not Intended for Diagnostic or Clinical Procedures.

2 Phospho-Specific Sandwich ELISA Kit Kit Includes Volume Solution Color Storage Capture Antibody Pre-Coated Microplate/Strip 96 tests 4 C Phospho-Specific Biotin-Conjugated Detection Antibody lyophilized Green Cap 4 C Avidin-conjugated HRP 10ul Blue Cap 4 C One-Step TMB Substrate 11ml Brown 4 C Stop Solution 11ml Colorless 4 C Adhesive plate Sealers 3 sheets 4 C Wash Buffer (10X) 2X20ml Colorless 4 C Detection Antibody Diluent 15ml Colorless 4 C Assay diluent 15ml Colorless 4 C Species Cross-Reactivity HeLa, HCT-116, THP-1, MD-MAB-231, Jurkat, Molt4 have been tested. Phospho-specific Sandwich ELISA can detect the phospho-specific proteins from lysate concentration range from 0.01 mg/ml to 1.20 mg/ml depending on different cell types or treatments. Reagent and Material Preparation Please read the following steps before you start the experiment. 1. Bring all microwell strips or 96-well microplateto room temperature before use. 2. Prepare 1X Wash Buffer by diluting 10X wash buffer (included in each Phospho-Sandwich ELISA Kit) in purified water. 3. Prepare Phospho-Specific Biotin-Conjugated Detection Antibody by reconstituting the lyophilized antibody with 10ml of Detection Antibody Diluent. 4. Prepare Avidin-Conjuageted HRP by diluting avidin-conjugated HRP in Blue Cap tube with Assay diluent, the dilution factor is 1:4000. Cell Lysate and Antibody Incubation 1. (Optional for Breakable Strips) After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed and stored at 4 C immediately. 2. (Optional) Dilute the sample or cell lysate in Assay Diluent (supplied in each Phospho-Sandwich ELISA Kit). Mix and vortex for a few seconds. Dilution factor and cell lysate final concentration varies depending on individual target molecule of interest. Cell lysate can be diluted up to 1:1 for test. The 1

3 individual data sheet for each kit provides detailed information regarding an appropriate dilution factor for lysates, lysate pretreatment, and kit assay results. 3. Add 100 µl of each diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hours at 25 C with gentle shaking. Alternatively, the plate can be incubated overnight at 4 C, which gives the best detection of target protein. 4. Gently remove the tape and wash wells: a. Flip the plate and discard the liquid into a receptacle. b. Wash 4 times with 1X Wash Buffer, 300 µl each time for each well, each wash for 5mins with gentle shaking. c. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well. Note: Do not allow wells to completely dry at any time. d. Clean the underside of all wells with a lint-free tissue. 5. Add 100 µl of Detection Antibody (Biotin-conjugated antibody) diluted in Detection Antibody Diluent torelevant wells. Seal with tape and incubate the plate for 1.5 hour at 25 C. 6. Repeat wash procedure as shown in Step 4 under Cell Lysate and Antibody Incubation. 7. Add 100 µl of Avidin-conjugated HRP diluted in Assay Diluent to each well. Seal with tape and incubate the plate for 30 minutes at 25 C. 8. Repeat wash procedure as shown Step 4 under Cell Lysate and Antibody Incubation. 9. Add 100 µl of TMB Substrate to each well. Seal with tape and incubate the plate for color development at 25 C up to 30mins. 10. Add 100 µl of STOP Solution to each well. Shake gently for a few seconds. 11. Read the plate at 450nm. NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution. a. Visual Determination - Read within 30 minutes after adding STOP Solution. b. Spectrophotometric Determination - Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 minutes after adding STOP Solution. 2

4 Phospho-p53 (S9) Sandwich ELISA Kit Description ImmunoWay Biotechnology - provided Phospho-p53 (S9) Sandwich ELISA Kit is a solid phase enzyme linked immunosorbent assay kit that detects endogenous phosphorylated p53 at the site of S9. p53 specific antibody has been coated onto the microwells. The microwells have been pre-blocked and stabilized. Another blocking is optional. After lysate incubation, p53 (phospho- and non-phospho) is captured by the coated capture antibody. After extensive washing, biotin-conjugated Phospho-p53 (S9) antibody is added for the detection of serine phosphorylation at the site of S9 in the captured p53 protein. Avidin-linked HRP is thereafter used to recognize the bound detection antibody. HRP substrate (One step TMB) is added for final color development and acidic stop solution is added. The optical density value of the developed yellow color is proportional to the quantity of p53 phosphorylated at S9. Background p53 is a transcription factor and major tumor suppressor that plays a major role in regulating cellular responses to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. p53 is modified post-translationally at multiple sites. DNA damage induces phosphorylation of p53 at S15, S20 and S37, reducing its interaction with the oncoprotein MDM2. MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation. Phosphorylated by many kinases including Chk2 and Chk1 at S20, enhancing its tetramerization, stability and activity. The phosphorylation by CAK at S392 is increased in human tumors and has been reported to influence the growth suppressor function, DNA binding and transcriptional activation of p53. Phosphorylation of p53 at S46 regulates the ability of p53 to induce apoptosis. The acetylation of p53 appears to play a positive role in the accumulation of p53 during the stress response. Following DNA damage, p53 becomes acetylated at K382, enhancing its binding to DNA. Deacetylation of p53 can occur through interaction with SIRT1, a deacetylase that may be involved in cellular aging and the DNA damage response. p53 regulates the transcription of a set of genes encoding endosomal proteins that regulate endosomal functions. These include STEAP3 and CHMP4C, which enhance exosome production, and CAV1 and CHMP4C, which produce a more rapid endosomal clearance of the EGFR from the plasma membrane. DNA damage regulates a p53-mediated secretory pathway, increasing the secretion of some proteins such as Hsp90, SERPINE1, SERPINB5, NKEF-A, and CyPA, and inhibiting the secretion of others including CTSL and IGFBP-2. Two alternatively spliced human isoforms have been reported. Isoform 2 is expressed in quiescent lymphocytes. Seems to be non-functional. May be produced at very low levels due to a premature stop codon in the mrna, leading to nonsense-mediated mrna decay. Specificity/Sensitivity 3

5 ImmunoWay Biotechnology - provided Phospho-p53 (S9) Sandwich ELISA Kit detects p53 when phosphorylated at S9. As shown in Figure 1, the gradient dependent detection of Phospho-p53 (S9) is achieved using Phospho-p53 (S9) Sandwich ELISA Kit. Protein Concentration (ug/ml) Absorbance OD 450nm Figure 1 The relationship between the protein concentration of HeLa lysate (ug/ml) and absorbance at 450nm is shown. Cells were treated with serum starvation for 5 days. 4