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QIA03 p53 ELISA Kit (Mutant Selective)

p53 ELISA Kit (Mutant Selective) MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
QIA03
  
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      Overview

      Replacement Information
      Description
      OverviewSuitable for the quantitative determination of mutant p53 protein. The antibodies utilized in this assay kit react with an epitope exposed on human and most mammalian mutant p53 proteins but not on wild-type p53, thus making the assay mutant-selective.
      Catalogue NumberQIA03
      Brand Family Calbiochem®
      Materials Required but Not Delivered• 2-20 µl, 20-200 µl and 200-1000 µl precision pipettors with disposable tips • Wash bottle or multi-channel dispenser for washing • 500 ml graduated cylinder • Deionized H2O • Spectrophotometer capable of measuring absorbance in 96 well plates at a wavelength of 405 nm • Plate sealers
      References
      ReferencesGannon, J.V., et al. 1990. EMBO J. 9, 1595. Lane, D.P., and Benchimol, S. 1990. Genes Dev. 4, 1. Werness, B.A., et al. 1990. Science 248, 76. Finlay, C A., et al. 1989. Cell 57, 1083. Pinhasi Kimhi, O., et al. 1986. Nature 320, 182. Sarnow, P., et al. 1982. Cell 28, 387. Lane, D.P., and Crawford, L.V. 1979. Nature 278, 261.
      Product Information
      Detection methodColorimetric
      Form36 or 42 Tests
      Format96-well plate
      Kit containsPre-Coated Plate with Removable Strip Wells, Lyophilized Standard, Sample Diluent, p53 Reporter Antibody, Peroxidase Conjugate, Substrate, Wash Buffer, Assay Buffer, Adhesive Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Assay range250-4000 pg/ml
      Assay timeOvernight
      Sample TypeCell extracts, serum, plasma, or other fluids
      Physicochemical Information
      Sensitivity250 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® p53 Mutant Selective ELISA Kit is a non isotopic immunoassay for the in vitro quantitation of mammalian (including human) mutant p53 protein in cell extracts, serum, plasma, or other fluids.
      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 at at 4°C. All of the reagents included with the mutant p53 assay have been tested for stability. Assay Buffer should not be used if cloudiness or solid matter is present. A small amount of precipitate may form during storage. This precipitate must be removed by centrifugation prior to use. Substrate solutions A and B should be colorless and remain colorless after being mixed (prior to addition to plate).
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsPre-Coated Plate with Removable Strip Wells, Lyophilized Standard, Sample Diluent, p53 Reporter Antibody, Peroxidase Conjugate, Substrate, Wash Buffer, Assay Buffer, Adhesive Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA03 0

      Documentation

      p53 ELISA Kit (Mutant Selective) Certificates of Analysis

      TitleLot Number
      QIA03

      References

      Reference overview
      Gannon, J.V., et al. 1990. EMBO J. 9, 1595. Lane, D.P., and Benchimol, S. 1990. Genes Dev. 4, 1. Werness, B.A., et al. 1990. Science 248, 76. Finlay, C A., et al. 1989. Cell 57, 1083. Pinhasi Kimhi, O., et al. 1986. Nature 320, 182. Sarnow, P., et al. 1982. Cell 28, 387. Lane, D.P., and Crawford, L.V. 1979. Nature 278, 261.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      p53 And Related Products
      User Protocol

      Revision08-October-2008 JSW
      Form36 or 42 Tests
      Format96-well plate
      Detection methodColorimetric
      Speciesmammalian
      StorageUpon arrival store the entire contents of the at at 4°C. All of the reagents included with the mutant p53 assay have been tested for stability. Assay Buffer should not be used if cloudiness or solid matter is present. A small amount of precipitate may form during storage. This precipitate must be removed by centrifugation prior to use. Substrate solutions A and B should be colorless and remain colorless after being mixed (prior to addition to plate).
      Intended useThe Calbiochem® p53 Mutant Selective ELISA Kit is a non isotopic immunoassay for the in vitro quantitation of mammalian (including human) mutant p53 protein in cell extracts, serum, plasma, or other fluids.
      Backgroundp53 is a nuclear phosphoprotein discovered and identified in SV40 transformed cells when it was found to co-immunoprecipitate with the SV40 large T antigen. It has since been shown that the adenovirus E1B protein and Types 16 and 18 papilloma virus E6 proteins also bind to p53 suggesting that these DNA tumor viruses target the same host protein in their transformation processes. In its non-mutated or "wild type" state, p53 is considered to be an anti oncogene product functioning as a negative regulator of cell division and found intracellularly in normal cells and tissues at very low levels. Wild type p53 is conserved across a wide variety of species including man, mouse, rat and frog. When point mutations occur in a number of highly conserved sequences of the p53 gene, a mutant protein is expressed exhibiting characteristics of an activated oncogene product. Mutant p53 protein is present at high levels in a high percentage of virtually all classes of human tumors and in most tumor cell lines growing in culture. Mutant p53 proteins have a half life of ~24 h as compared to wild type p53 that has a half life of about 20 min. This extended half life allows mutant p53 proteins to accumulate to detectable levels in those tumors and tumor cell lines associated with an activated p53 oncogene. In contrast, wild type p53 does not accumulate and is not easily detected. Finally, mutant and wild type p53 proteins can be distinguished on the basis of differential reactivity with monoclonal antibodies. Antibody reactive with a conformationally sensitive epitope, located approximately between amino acids 155 and 215, reacts with most mammalian mutant p53 proteins but not with wild type p53, as long as the proteins have not been denatured. Denaturation may alter the conformation of the p53 protein exposing different epitopes and altering the reactivity of the protein with specific antibodies. The antibodies utilized in this assay kit react with an epitope exposed on human and most mammalian mutant p53 proteins but not on wild type p53 thus making the assay mutant selective.
      Principles of the assayThe p53 Mutant Selective ELISA Kit is a "sandwich" enzyme immunoassay employing both mouse monoclonal and rabbit polyclonal antibodies. Monoclonal antibodies, specific for the majority of undenatured mammalian mutant p53 proteins, have been immobilized onto the surface of the plastic wells provided in the kit. The sample to be assayed is pipetted into the wells and allowed to incubate for a period of time, during which any mutant p53 present binds to the capture antibody. Unbound material is washed away, and rabbit polyclonal reporter antibody is added. This antibody also recognizes mutant mammalian p53 proteins, and will bind to any mutant p53 that has been retained by the capture antibody. The reporter antibody, in turn, is bound by horseradish peroxidase conjugated goat anti rabbit immunoglobulin G (IgG). The horseradish peroxidase catalyses the conversion of the chromogenic substrate 2,2' azino di [3 ethyl benzthiazoline sulfonate] (ABTS) from a colorless solution to a blue/green solution, the intensity of which is proportional to the amount of mutant p53 protein bound to the plate. The colored reaction product is quantified using a spectrophotometer. Quantitation is achieved by the construction of a standard curve using dilutions of a known concentration of purified, recombinant human mutant p53 (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of mutant p53 with that obtained from the standard curve, the concentration of mutant p53 in the sample can be determined.
      Materials providedSamples and standards should be assayed in duplicate. A standard curve must be performed each time samples are analyzed. The mutant p53 ELISA provides sufficient reagents to run two standard curves, and 42 samples (if assayed all at once using one standard curve), or 36 samples (if assayed on two separate occasions using two standard curves). • 96-Well Assay Plate coated with mutant p53 monoclonal antibody: 96 removable wells • Mutant p53 Standards: two individual vials containing pre-measured, lyophilized recombinant human mutant p53. Reconstitute, with Sample Diluent, as described on the vial to yield a 4 ng/ml solution. Reconstituted standards should be discarded after one use. • p53 Reporter Ab: (0.5 ml) 20 fold concentrated solution of rabbit p53 antibody dissolved in Dilution Buffer. Contains 0.02% 2-Chloroacetamide. • Peroxidase Conjugate: (0.2 ml) 100 fold concentrated solution of horseradish peroxidase-conjugated to goat anti rabbit IgG. Contains 0.02% 2-Chloroacetamide. • Peroxidase substrate: (5 ml) Reagent A (ABTS) (5 ml) Reagent B (H₂O₂) Mix equal volumes of reagent A with reagent B just prior to use. • Sample Diluent: (25 ml) Phosphate buffered saline (PBS) containing bovine serum albumin, a surfactant, and 0.02% 2-Chloroacetamide. • Assay Buffer: (25 ml) Phosphate buffered saline containing surfactant, protein stabilizers and 0.02% 2-Chloroacetamide. • Wash Buffer: (50 ml) 10 fold concentrated solution of PBS and surfactant. Contains 0.2% 2-Chloroacetamide. • Plate Sealers: Used to cover plates during incubations.
      Materials Required but not provided• 2-20 µl, 20-200 µl and 200-1000 µl precision pipettors with disposable tips • Wash bottle or multi-channel dispenser for washing • 500 ml graduated cylinder • Deionized H2O • Spectrophotometer capable of measuring absorbance in 96 well plates at a wavelength of 405 nm • Plate sealers
      Precautions and recommendations• Store all components at 4°C. Do not expose reagents to excessive light. Do not freeze any component. • Use only the wells provided with the kit. • Do not mix reagents from different kits. • The buffers and reagents used in this kit contain 2-Chloroacetamide. Care should be taken to prevent direct contact with these products. • Do not mouth pipette or ingest any of the reagents. • Do not smoke, eat, or drink when performing the assay or in areas where samples/reagents are handled. • Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
      PreparationSamples should be free of any particulate matter. Remove any flocculent material by centrifugation and/or filtration of the samples prior to use. Fluid samples (e.g. serum, plasma, etc.) may be analyzed without further treatment. Samples found to contain greater than 4 ng/ml of mutant p53 must be diluted with Sample Diluent, so that the p53 concentration falls within the range spanned by the standard curve, and re assayed. For instance, a sample containing an unknown concentration of mutant p53 could be simultaneously assayed undiluted, diluted 1:10 and 1:100 in Sample Diluent. This would permit an accurate determination of the mutant p53 concentration in the sample over a range of 0.25 ng/ml to 400 ng/ml. To measure the concentration of mutant p53 in cells, an extract must first be prepared. Numerous extraction protocols can be used. The following protocol has been shown to work with a number of cultured cell lines. It is provided as an example of a whole cell extraction procedure and should not be construed as necessarily being the method of choice. It is strongly recommended that individual users experiment with extraction procedures that work best for any particular cell line or tissue under study. Whole Cell Extraction Procedure: Pellet cells by centrifugation (1000 X g, for 10 min at 4°C) then wash 3 times with 20 to 30 cell pellet volumes of ice cold PBS. Pellet cells and discard PBS wash. Add 5 cell pellet volumes of ice cold swelling buffer (20 mM Tris/HCI, 5 mM EDTA, 1 mM PMSF, pH 8) and incubate on ice for 30 min. Gently resuspend cells every 10 min. Add non ionic detergent (e.g. Tween®-20 detergent) to a final concentration of 1% (v/v) and continue to incubate on ice for an additional 30 min. Resuspend cells every 10 min. Add NaCl to a final concentration of 0.5 M. Incubate for 15 min on ice. Resuspend every 5 min. Pellet at ~70,000 X g for 30 min at 4°C (e.g., 70.1 Ti at 30,000 RPM). Carefully collect the membrane free supernatant. Supernatant (whole cell extract) may be analyzed for mutant p53 concentration immediately, or stored frozen at 70°C for later analysis. Whenever possible, appropriate negative controls should be included.
      Detailed protocol

      Figure 1: Protocol Summary

      The p53 Mutant Selective ELISA Kit is provided with removable strips of wells so the assay can be carried out on two separate occasions. Since conditions may vary, a standard curve MUST be determined each time the assay is performed. Both standards and unknown samples should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross contamination of reagents or samples. A. Remove the appropriate number of wells from the foil pouch and return any unused wells to the foil pouch, seal with tape, and store at 4°C. B. Prepare a working solution (1 X) of Wash Buffer by adding 50 ml of the 10X concentrated solution (provided), to 450 ml of deionized water. Mix well. C. Each time an assay is performed, reconstitute a Lyophilized Standard by carefully and accurately pipetting the amount of Sample Diluent described on the lyophilized p53 STANDARD vial to give a concentration of 4 ng/ml. Let the reconstituted standard sit for 15 min, with occasional swirling. Avoid excessive agitation of the standard. After reconstituting the p53 Lyophilized Standard it should be diluted with Sample Diluent. Obtain six tubes and label them 4, 2, 1, 0.5, 0.25, and 0 ng/ml. Add 300 µl of Sample Diluent into each tube except the first tube (4 ng/ml), which gets "undiluted" reconstituted standard. Remove 600 µl from the original vial of lyophilized material and add it to the first tube. Remove 300 µl from the first tube (4 ng/ml) and add it to the second tube (2 ng/ml) and mix gently. Repeat this procedure until you reach the fifth tube (0.25 ng/ml). The last tube (0 ng/ml) should just be Sample Diluent. Reconstituted standards should be discarded after one use. D. Wash wells once using 300 µl of 1 X Wash Buffer per well. E. Remove Wash Buffer by inverting the plate and tapping on paper towels. F. Add samples and each of the p53 standards (in duplicate) by pipetting 100 µl into appropriate wells using clean pipette tips for each sample. If sample dilution is necessary use the Sample Diluent supplied (See Sample Preparation). G. Cover wells with a plate sealer and incubate at 4°C overnight. H. Wash wells 4 times using 300 µl of 1 X Wash Buffer per well. I. Dilute sufficient p53 Reporter antibody 1:20 in Assay Buffer to provide 100 µl of 1X solution for each of the sample and standard wells, mix gently. (For example: add 50 µl to 950 µl of Assay Buffer to make 1 ml of working solution, sufficient for 10 wells). In order not to run short of material, it is recommended that 5-10% more working solution be prepared than is required for the number of wells being tested. J. Pipette 100 µl of the 1 X p53 Reporter Ab into each well, cover with a plate sealer and incubate at room temperature for 2 h. Discard any unused 1 X Reporter antibody. K. Wash wells 4 times using 300 µl of 1 X Wash Buffer per well. L. Dilute a sufficient amount of the Peroxidase Conjugate 1:100 in Assay Buffer to provide 100 µl of 1X solution for each of the sample and standard wells, mix gently. (For example: add 10 µl to 990 µl of Assay Buffer to make 1 ml of working solution). M. Pipette 100 µl of the 1X Peroxidase Conjugate into each well, cover with a plate sealer and incubate at room temperature for 1 h. Discard any unused 1 X Peroxidase Conjugate. N. Wash wells 4 times using 300 µl of 1 X Wash Buffer per well. O. Prepare substrate solution by mixing together equal amounts of substrate solutions A and B. Pipette 100 µl of prepared substrate into each well. Substrate solution must be used within 30 min of preparation. Discard any unused prepared substrate solution. P. Incubate the wells at room temperature for 30 min, gently mix the plate and read the absorbance of each well on a spectrophotometric plate reader at a wavelength of 405 nm.
      CalculationsA. Prepare a standard curve by plotting the average absorbance (A405) versus the concentration (ng/ml) for each dilution of the mutant p53 standard. Fit the best line to the points. This can be done manually or for greater precision, by using a curve-fitting program. A typical standard curve is shown below:

      Figure 2: Calibration Curve

      To determine the mutant p53 concentration in each of the samples, first calculate the average absorbance value for each set of duplicates. This value can then be entered into the linear regression equation to yield the mutant p53 concentration of the sample. Alternatively, locate the point at which the average absorbance value intersects the standard curve and draw a vertical line down to the X axis. The mutant p53 concentration can be read directly from the standard values on the X axis (i.e., if the vertical line falls midway between the 0.5 ng/ml and the 1 ng/ml standards, then the sample contains 0.75 ng/ml mutant p53. For samples that have been diluted, the mutant p53 concentration must be multiplied by the dilution factor (i.e., if the sample was diluted five fold, then the mutant p53 value obtained from the standard curve must be multiplied by five).
      Sensitivity250 pg/ml
      Sensitivity NotesThe Calbiochem® p53 Mutant Selective ELISA Kit will detect 0.25 ng/ml of purified human recombinant mutant p53 diluted in Sample Diluent with a signal that is approximately twice background absorbance. The minimum concentration of mutant p53 that can be detected is 0.05 ng/ml.
      Assay Range250-4000 pg/ml
      RecoveryMutant p53 proteins have been shown to associate with the heat shock protein 70 family of proteins. Additional less well understood protein protein interactions between mutant p53 and other cellular or serum proteins may also occur. Such interactions could potentially mask antigenic epitopes on the p53 protein and thus interfere with the mutant p53 assay's ability to detect mutant p53 when bound to these proteins. When a known amount of purified recombinant human mutant p53 (up to 4 ng/ml) was added to p53 free cell lysates or sera, recovery of the added p53 varied from 10% to 60% depending on the amount of p53 added.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc. Tween® is a registered trademark of ICI Americas, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.