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CBA009 PhosphoDetect™ p53 (pSer¹⁵) ELISA Kit

PhosphoDetect™ p53 (pSer¹⁵) ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
CBA009
  
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Colorimetric
      Description
      Overview

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      This product has been discontinued.



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      Detects p53 when phosphorylated on Ser15 in human cell lysates. Does not react with mouse or rat. p53 is phosphorylated on Ser15 by ATM, ATR, and DNA protein kinase leading to reduced interactions with its negative regulator, MDM2, and accumulation of p53.
      Catalogue NumberCBA009
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Plate reader capable of measurement at or near 450 nm
      • Calibrated adjustable precision pipettes with disposable plastic tips, a manifold multi-channel pipette is helpful for large assays
      • Cell Lysis Buffer, see page 3
      • Deionized or distilled H2O
      • Automated or manual plate washer such as a squirt bottle or manifold dispenser
      • Linear, log-log, or semi-log graph paper
      • Glass or plastic tubes for diluting and aliquoting standard
      • Absorbent paper towels
      • Calibrated beakers and graduated cylinders in various sizes
      References
      ReferencesHirao, A., et al. 2000. Science 287, 1824.
      Prives, C. 1999. J. Pathol. 187, 122.
      Shieh, S.Y., et al. 1999. EMBO J. 18, 1815.
      Prokocimer, M., et al. 1998. Hum. Mutat. 12, 4.
      Steele, R.J., et al. 1998. Br. J. Surg. 85, 1460.
      Hung, J., et al. 1997. Acta Orthop. Scand. Suppl. 273, 68.
      Levine, A.J. 1997. Cell 88, 323.
      Milczarek, G.J., et al. 1997. Life Sci. 60, 1.
      Milner, J. 1997. Pathol. Biol. (Paris) 45, 797.
      Shieh, S.Y., et al. 1997. Cell 91, 325.
      Shaw, P.H. 1996. Pathol. Res. Pract. 192, 669.
      Meek, D.W. 1994. Semin. Cancer Biol. 5, 203.
      El-Deiry, W.S., et al. 1993. Cell 75, 817.
      Lane D.P. 1992. Nature 358, 15.
      Tominaga, O., et al. 1992. Crit. Rev. Oncog. 3, 257.
      Hollstein, M., et al. 1991. Science 253, 49.
      Product Information
      Unit of DefinitionOne unit is defined as the amount of p53 (pSer¹⁵) derived from approximately 11,000 CCRF/CEM cells treated with 5 nM actinomycin D for 20 hours.
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, p53 Phospho-Ser¹⁵ Standard, Diluents, Detector Antibody, Secondary Antibody, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Applications
      Biological Information
      Assay range1-100 units/ml
      Assay time4 h
      Sample TypeCell lysates
      Physicochemical Information
      Sensitivity≤0.9 units/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, p53 Phospho-Ser¹⁵ Standard, Diluents, Detector Antibody, Secondary Antibody, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalog Number GTIN
      CBA009 0

      Documentation

      PhosphoDetect™ p53 (pSer¹⁵) ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA009

      References

      Reference overview
      Hirao, A., et al. 2000. Science 287, 1824.
      Prives, C. 1999. J. Pathol. 187, 122.
      Shieh, S.Y., et al. 1999. EMBO J. 18, 1815.
      Prokocimer, M., et al. 1998. Hum. Mutat. 12, 4.
      Steele, R.J., et al. 1998. Br. J. Surg. 85, 1460.
      Hung, J., et al. 1997. Acta Orthop. Scand. Suppl. 273, 68.
      Levine, A.J. 1997. Cell 88, 323.
      Milczarek, G.J., et al. 1997. Life Sci. 60, 1.
      Milner, J. 1997. Pathol. Biol. (Paris) 45, 797.
      Shieh, S.Y., et al. 1997. Cell 91, 325.
      Shaw, P.H. 1996. Pathol. Res. Pract. 192, 669.
      Meek, D.W. 1994. Semin. Cancer Biol. 5, 203.
      El-Deiry, W.S., et al. 1993. Cell 75, 817.
      Lane D.P. 1992. Nature 358, 15.
      Tominaga, O., et al. 1992. Crit. Rev. Oncog. 3, 257.
      Hollstein, M., et al. 1991. Science 253, 49.
      User Protocol

      Revision21-October-2008 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C.
      Backgroundp53 is the most commonly mutated gene in human cancer. Mutations and allele loss in the gene, located on chromosome 17p, are the most frequent alterations yet identified in human malignancies, with more than 500 mutations described. These mutations are found in various types of malignancies, hematologic as well as solid tumors. However, all the mutants are not necessarily equivalent in terms of biological activity. The p53 protein is highly conserved and expressed in normal tissues. Wild-type p53 is shown to be a sequence-specific transcription factor, directly interacting with various cellular and viral proteins. Studies demonstrate that intact p53 function is essential for the maintenance of the non-tumorogenic phenotype of cells. Thus, p53 plays a vital role in suppressing the development of cancer. The p53 tumor suppressor protein is important in the cellular response to DNA damage and other genomic aberrations. Cells exposed to DNA-damaging agents such as ionizing radiation, UV radiation, and chemical agents initiate a complex response that includes the inhibition of cell cycle progression until damage is repaired. If the DNA damage is beyond repair, cells may enter a prolonged state of arrest or undergo a programmed cell death, known as apoptosis, thereby maintaining genetic stability in the organism. In response to DNA damage, p53 is phosphorylated at multiple sites by several protein kinases. Phosphorylation of p53 at Ser15 by ATM, ATR, and DNAPK leads to a reduced interaction with its negative regulator, MDM2, and accumulation of p53 protein. Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its activity, tetramerization, and stability. Elevation of p53 protein induces the transcriptional activation of multiple genes, including p21waf1. p21waf1 interacts directly with cyclin-dependent kinases, important for cell cycle progression, thereby inhibiting their activity and resulting in cell cycle arrest.
      Principles of the assayThe Calbiochem® PhosphoDetect™ p53 (Ser¹⁵) ELISA kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for p53 (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing phospho-p53 (Ser¹⁵), control specimens, and unknowns, are pipetted into these wells. During the first incubation, the p53 antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for p53 phosphorylated at serine 15 is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized p53 protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase-labeled anti-rabbit IgG (anti-rabbit IgG-HRP) is added. This binds to the detection antibody to complete the four-member sandwich. After a third incubation and washing to remove all the excess anti-rabbit IgG-HRP, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of phospho-p53 (Ser¹⁵) present in the original specimen.
      Materials provided• Phospho-p53 (Ser¹⁵) Standard (Kit Component No. JA7820-1EA): 2 vials; lyophilized cell lysate from actinomycinD-treated CCRF/CEM cells; please refer to vial label for quantity and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA7821-25ML): 1 bottle, 25 ml; contains 15 mM sodium azide
      • Coated 96-Well Plate (Kit Component No. JA7822-1EA): 1 plate, 96-wells,Supplied as twelve 8-well strips coated with anti-p53 antibody
      • Detection Antibody (Kit Component No. JA7823-11ML): 1 bottle, 11 ml, rabbit anti-phospho-p53 (Ser¹⁵); contains 15 mM sodium azide
      • Secondary Antibody (Kit Component No. JA7824-125UL): 1 vial, 0.125 ml, 100X anti-rabbit IgG-Horseradish Peroxidase (HRP) concentrate containing 3.3 mM thymol
      • Secondary Antibody Diluent (Kit Component No. JA7824-25ML): 1 bottle, 25 ml; contains 3.3 mM thymol
      • Wash Buffer Concentrate (Kit Component No. JA7826-100ML): 1 bottle, 100 ml 25X wash buffer
      • HRP Substrate (TMB) (Kit Component No. JA7827-25ML): 1 bottle, 25 ml stabilized chromogen (Tetramethylbenzidine)
      • Stop Solution (Kit Component No. JA7828-25ML): 1 bottle, 25 ml
      • Plate Covers (Kit Component No. JA7829-1EA): 3 adhesive strips

      Note: Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state, and local regulations for disposal.
      Materials Required but not provided• Plate reader capable of measurement at or near 450 nm
      • Calibrated adjustable precision pipettes with disposable plastic tips, a manifold multi-channel pipette is helpful for large assays
      • Cell Lysis Buffer, see page 3
      • Deionized or distilled H2O
      • Automated or manual plate washer such as a squirt bottle or manifold dispenser
      • Linear, log-log, or semi-log graph paper
      • Glass or plastic tubes for diluting and aliquoting standard
      • Absorbent paper towels
      • Calibrated beakers and graduated cylinders in various sizes
      Precautions and recommendations1. When not in use, kit components should be stored at 4°C. All reagents should be warmed to room temperature before use.
      2. Plates should be allowed to come to room temperature before opening the foil bags. Once the desired number of strips has been removed, immediately reseal the bag and store at 4°C to maintain plate integrity.
      3. Samples should be frozen if not analyzed shortly after collection, avoiding multiple freeze-thaw cycles. Thaw samples completely and mix well prior to analysis.
      4. If particulate matter is present, centrifuge or filter prior to analysis.
      5. All standards, controls, and samples should be run in duplicate.
      6. Samples containing phospho-p53 (Ser15) protein extracted from cells should be diluted with Standard Diluent Buffer at least 1:10. This dilution is necessary to reduce the matrix effect of the Cell Lysis Buffer.
      7. When pipetting reagents, maintain a consistent order of addition from well to well. This ensures equal incubation times for all wells.
      8. Cover or cap all reagents when not in use.
      9. Do not mix or interchange different reagent lots from various kit lots.
      10. Read absorbances within 2 h of assay completion.
      11. In-house controls should be run with every assay. If control values fall outside pre-established ranges, the accuracy of the assay is questionable.
      12. All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Do not insert absorbent paper directly into the wells.
      13. Because TMB is light sensitive, avoid prolonged exposure to light. Avoid contact between TMB and metal, or color may develop.
      14. All blood components and biological materials should be handled as potentially hazardous. Follow precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.
      15. Washing Tips:
      a. Incomplete washing will adversely affect the test outcome. All washing must be performed with the Wash Buffer provided.
      b. Completely aspirate the liquid from all wells by gently lowering an aspiration tip into the bottom of each well. Take care not to scratch the inside of the well.
      c. After aspiration, fill the wells with at least 0.4 ml of Diluted Wash Buffer. Let soak for 15 to 30 s, then aspirate the liquid. If a squirt bottle is used, flood the plate with Diluted Wash Buffer, completely filling all wells. If using an automated washer, follow the operating instructions for the washing equipment. If possible, 30 s soak cycles should be programmed into the wash cycle. Repeat as directed in the assay procedure that follows. After the washing procedure, invert the plate and tap dry on absorbent towels.
      Preparation• Cell Lysis Buffer: • 10 mM Tris, pH 7.4 • 100 mM NaCl • 1 mM EDTA • 1 mM EGTA • 1 mM NaF • 20 mM Na4P2O7 • 2 mM Na3VO4 • 1% Triton® X-100 detergent • 10% glycerol • 0.1% SDS • 0.5% deoxycholate • 1 mM PMSF (stock is 0.3 M in DMSO) Protease inhibitor cocktail (Cat. No. 539134) should be used at a dilution of 1:100-1:300. Cell Lysis Buffer without protease inhibitors and PMSF is stable for 2-3 weeks at 4°C or for up to 6 months when aliquoted and stored at -20°C. When stored frozen, this buffer should be thawed on ice. Add protease inhibitors just prior to use. PMSF is very unstable and must be added just prior to use, even if added previously. • Cell Lysate Preparation: Note: Users should optimize the cell lysis procedure for their own applications. 1. Collect cells in PBS by centrifugation (non-adherent) or scraping from culture flasks (adherent). 2. Wash cells twice with cold PBS. 3. Remove and discard the supernatant and collect the cell pellet. This cell pellet may be frozen at -80°C and lysed at a later date. 4. Lyse the cell pellet in Cell Lysis Buffer for 30 min, on ice, vortexing at 10 min intervals. The volume of Cell Lysis Buffer depends on the number of cells in the cell pellet and the expression of phospho-p53 (Ser15). For example, 107 CCRF/CEM cells grown in RPMI plus 10% FBS and treated with 5 nM actinomycin D for 20 h can be extracted in 1 ml of Cell Lysis Buffer. Under these conditions, the use of 1-10 µl of the clarified cell lysate diluted to a volume of 100 µl per well in standard Diluent Buffer is sufficient for the detection of phospho-p53 (Ser15). 5. Transfer lysate to microcentrifuge tubes and centrifuge at 13,000 rpm for 10 min at 4°C. 6. Aliquot the clear lysate (supernatant) to clean microfuge tubes. Proceed to the sample treatment step below prior to assay. Lysates can be stored at -80°C. Avoid multiple freeze/thaw cycles.
      Reagent preparation• Phospho-p53 (Ser15) Standard: Note: The Phospho-p53 (Ser15) Standard is designated in units/ml. One unit of standard is equivalent to the amount of phospho-p53 (Ser15) derived from approximately 11,000 CCRF/CEM cells treated with 5 nM actinomycin D for 20 h. 1. Reconstitute Phospho-p53 (Ser15) Standard with the appropriate volume of Standard Diluent Buffer as instructed on the standard vial label. Swirl or mix gently and allow to sit for 10 min to ensure complete reconstitution. Label as 100 units/ml phospho-p53 (Ser15). Use standard within 1 h of reconstitution. 2. Add 0.25 ml of Standard Diluent Buffer to each of 6 tubes labeled 50, 25, 12.5, 6.25, 3.12 and 1.6 units/ml phospho-p53 (Ser15). 3. Make serial dilutions of the standard as described in the following table. Mix thoroughly between steps.

      Table 1: Phospho-p53 (Ser15) Standard Dilutions

      Remaining reconstituted standard should be discarded or frozen at -80°C for further use. Standard can be frozen and thawed one time only without loss of immunoreactivity. • Secondary Antibody Working Solution: Note: The Secondary Antibody is a viscous solution containing 50% glycerol. To ensure accurate dilution, allow the Secondary Antibody to reach room temperature. Mix gently and pipette slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper. Dilute 10 µl of the Secondary Antibody with 1 ml Secondary Antibody Diluent for each 8-well strip used in the assay. Label as Secondary Antibody Working Solution. For example:

      Table 2: Secondary Antibody Dilutions

      2. Return the unused Secondary Antibody to the refrigerator. • 1X Wash Buffer: Allow the Wash Buffer Concentrate to reach room temperature and mix to ensure that any precipitated salts have redissolved. To prepare 1X Wash Buffer dilute 1 volume of the Wash Buffer Concentrate with 24 volumes of deionized water (for example, 50 ml may be diluted up to 1.25 liters; 100 ml may be diluted up to 2.5 liters). Store both the Wash Buffer Concentrate and the 1X Wash Buffer in the refrigerator. The 1X Wash Buffer should be used within 14 days.
      Detailed protocolNote: Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use. A standard curve must be run with each assay.
      1. Determine the number of 8-well strips needed for the assay. Insert these in the frame(s) for use. Re-bag extra strips and frame; store these at 4°C for future use.
      2. Add 100 µl of the Standard Diluent Buffer to zero wells. Well(s) reserved for chromogen blank should be left empty.
      3. Add 100 µl of standards, samples, or controls to the appropriate wells. Samples prepared in Cell Lysis Buffer must be diluted of at least 1:10 in Standard Diluent Buffer (higher dilutions such as 1:25 or 1:50 may be optimal). Note: The Cell Lysis Buffer and final dilution chosen should be optimized for each experimental system. Tap gently on side of plate to thoroughly mix.
      4. Cover the wells with a Plate Cover and incubate for 2 h at room temperature or overnight at 4°C.
      5. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times as described in washing section above.
      6. Add 100 µl Detection Antibody to each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
      7. Cover the wells with a Plate Cover and incubate for 1 h at room temperature.
      8. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times as described above.
      9. Add 100 µl Secondary Antibody Working Solution to each well except the chromogen blank(s).
      10. Cover the wells with a Plate Cover and incubate for 30 min at room temperature.
      11.Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times as described previously.
      12. Add 100 µl HRP Substrate (TMB) to each well. The liquid in the wells will begin to turn blue.
      13. Incubate for 30 min at room temperature in the dark. Note: Do not cover the plate with aluminum foil or metalized mylar. The incubation time for the HRP Substrate (TMB) is often determined by the plate reader used. Many plate readers have the capacity to record a maximum absorbance (Abs) of 2.0. The absorbance values should be monitored and the substrate reaction stopped before the absorbance of the positive wells exceeds the limits of the instrument. The absorbance values at 450 nm can be read only after the stop solution has been added to each well. If using a reader that records only to 2.0 absorbance, stopping the assay after 20 to 25 min is suggested.
      14. Add 100 µl Stop Solution to each well. Tap the side of plate gently to mix. The solution in the wells should change from blue to yellow.
      15. Read the absorbance of each well at 450 nm, having blanked the plate reader against a chromogen blank containing 100 µl each HRP Substrate (TMB) and Stop Solution. Read the plate within 2 h after adding the Stop Solution.
      16. Plot the absorbance of the standards against the standard concentration. Optimally, the background absorbance may be subtracted from all data points, including standards, unknowns and controls, prior to plotting. Draw the best smooth curve through these points to construct the standard curve. If using curve fitting software, the four parameter algorithm provides the best curve fit.
      17. Read the phospho-p53 (Ser15) concentrations for unknown samples and controls from the standard curve plotted in step 16. Multiply the values obtained for the samples by the dilution factor to correct for the dilution in step 3. Samples producing signals higher than the highest standard (100 units/ml) should be further diluted in Standard Diluent Buffer and re-analyzed, multiplying the concentration by the appropriate dilution factor.
      Standard curveThe following data was obtained for the various standards over the range of 0 to 100 units/ml phospho-p53 (Ser¹⁵).

      Table 3: Example Data
      Limitations of the assayDo not extrapolate the standard curve beyond the 100 units/ml standard point; the dose-response is non-linear in this region and accuracy is questionable. Dilute samples greater than 100 units/ml with Standard Diluent Buffer and re-analyze and multiply the results by the appropriate dilution factor. The influence of various lysis buffers has not been thoroughly investigated. The rate of degradation of native p53 or dephosphorylation of phospho-p53 (Ser¹⁵) in various matrices has not been investigated. Although p53 degradation or dephosphorylation of phospho-p53 (Ser¹⁵) in the Cell Lysis Buffer used in this protocol has not been observed, the possibility cannot be excluded.
      Sensitivity≤0.9 units/ml
      Sensitivity NotesThe analytical sensitivity of this assay is less than 0.9 units/ml of human phospho-p53 (Ser¹⁵). This was determined by adding two standard deviations to the mean absorbance obtained when the zero standard was assayed 30 times. The sensitivity of this ELISA as compared to immunoblotting using known quantities of phospho-p53 (Ser¹⁵), (below), shows that the sensitivity of the ELISA is approximately 8X greater than that of immunoblotting. The bands shown in the immunoblot were developed using rabbit anti-phospho-p53 (Ser¹⁵) and an alkaline phosphatase conjugated anti-rabbit IgG followed by chemiluminescent detection.

      Figure 1: Sensitivity
      Assay Range1-100 units/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known phospho-p53 (Ser15) concentration were assayed in replicates of 12 to determine precision within an assay.


      Table 5: Inter-Assay Precision

      Samples were assayed 36 times in multiple assays to determine precision between assays.
      RecoveryThe recovery of phospho-p53 (Ser¹⁵) added to Jurkat cell lysate (a p53 deficient cell line) (adjusted to 10 µg/ml total protein) averaged 102% when diluted in Standard Diluent Buffer.
      Parallelism

      Figure 2: Human p53 (pSer15): Parallelism
      LinearityCCRF/CEM cells were grown in cell culture medium containing 10% fetal calf serum, treated with 5 nM actinomycin D for 20 h and lysed with cell extraction buffer. This lysate was diluted in standard diluent buffer over the range of the assay and measured for phospho-p53 (Ser¹⁵). Linear regression analysis of sample values versus the expected concentration yielded a correlation coefficient of 0.99.

      Table 6: Linearity of Dilution
      Specificity

      Figure 3: Peptide Competition: Specificity

      The specificity of this assay for phosphorylated phospho-p53 (Ser15) was confirmed by peptide competition. The data shows that the phospho-peptide containing the phosphorylated serine 15 blocks the ELISA signal.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Triton® is a registered trademark of Dow Chemical Company
      Interactive Pathways™ and PhosphoDetect™ are trademarks of EMD Chemicals, Inc.

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