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CBA008 PhosphoDetect™ p38 MAP Kinase (pThr¹⁸⁰/pTyr¹⁸²) ELISA Kit

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
CBA008
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      Detects the dually phosphorylated form of p38 MAP kinase at Thr180 and Tyr182.This activation occurs in response to a variety of extracellular stimuli including osmotic shock, inflammatory cytokines, LPS, and UV light. Although this kit was designed for human samples, it cross-reacts with mouse and monkey.
      Catalogue NumberCBA008
      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
      • Lysis Buffer
      • Deionized or distilled H2O
      • PBS
      • 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
      ReferencesGe, B., et al. 2002. Science 295, 1291.
      Browning, D. D., et al. 2000. J. Biol. Chem. 275, 2811.
      Ono, K., and Han, J. 2000. Cell Signal. 12, 1.
      Hale, K. K., et al. 1999. J. Immunol. 162, 4246.
      Wang, X.S., et al. 1997. J. Biol. Chem. 272, 23668.
      Han, J., et al. 1996. J. Biol. Chem. 271, 2886.
      Zervos, A.S., et al. 1995. Proc. Natl. Acad. Sci. USA 92, 10531.
      Han, J., et al. 1994. Science 265, 808.
      Lee, J.C., et al. 1994. Nature 372, 739.
      Ershler, M., et al. 1993. Gene 124, 305.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsp38 MAP Kinase Antibody Coated 96-Well Plate, p38 MAP Kinase (pThr¹⁸⁰/pTyr¹⁸²) Standard, Standard Diluent Buffer, Detection Antibody, Secondary Antibody, Secondary Antibody Diluen, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Applications
      Biological Information
      Assay range1.6-100 U/ml
      Assay time4 h
      Sample TypeCell lysates
      Physicochemical Information
      Sensitivity≤0.8 U/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 21/22-36/38

      Harmful in contact with skin and if swallowed.
      Irritating to eyes and skin.
      S PhraseS: 36/37-45

      Wear suitable protective clothing and gloves.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Harmful
      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 containsp38 MAP Kinase Antibody Coated 96-Well Plate, p38 MAP Kinase (pThr¹⁸⁰/pTyr¹⁸²) Standard, Standard Diluent Buffer, Detection Antibody, Secondary Antibody, Secondary Antibody Diluen, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA008 0

      Documentation

      PhosphoDetect™ p38 MAP Kinase (pThr¹⁸⁰/pTyr¹⁸²) ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA008

      References

      Reference overview
      Ge, B., et al. 2002. Science 295, 1291.
      Browning, D. D., et al. 2000. J. Biol. Chem. 275, 2811.
      Ono, K., and Han, J. 2000. Cell Signal. 12, 1.
      Hale, K. K., et al. 1999. J. Immunol. 162, 4246.
      Wang, X.S., et al. 1997. J. Biol. Chem. 272, 23668.
      Han, J., et al. 1996. J. Biol. Chem. 271, 2886.
      Zervos, A.S., et al. 1995. Proc. Natl. Acad. Sci. USA 92, 10531.
      Han, J., et al. 1994. Science 265, 808.
      Lee, J.C., et al. 1994. Nature 372, 739.
      Ershler, M., et al. 1993. Gene 124, 305.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protein Kinase Assay and Detection Kits Brochure
      User Protocol

      Revision21-October-2008 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, monkey, mouse
      StorageUpon arrival, store the entire contents of the kit at 4°C.
      Backgroundp38 MAP kinase (MAPK), also known as RK (CDC2-related protein kinase) or CSBP (cytokine suppressive anti-inflammatory drug binding protein), is the mammalian homologue of the yeast HOG kinase (high osmolarity glycerol response kinase). There are at least three distinct mitogen-activated protein kinase (MAP kinase) signaling modules which mediate extracellular signals into the nucleus to turn on the responsive genes in mammalian cells, including extracellular mitogen-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK, also called stress-activated protein kinase, SAPK), and p38 kinase. The p38 signaling transduction pathway plays an essential role in regulating many cellular processes including inflammation, cell differentiation, cell growth and death. p38 MAP kinase is activated in response to a variety of extracellular stimuli including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), anisomycin, UV light, and growth factors. The activation of p38 MAP kinase is mediated by several upstream kinases including MAP kinase-kinase 3 (MKK3), MAP kinase-kinase 6 (MKK6), and MAP kinase-kinase 4 (MKK4, also known as SEK1 and JNKK1). These kinases phosphorylate p38 at Thr180 and Tyr182 in the TGY motif, resulting in p38 activation. Recently MAP kinase kinase-independent activation of p38 was shown to involve the interaction of p38α with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38α. Various targets of p38 have been identified including transcription factors ATF-2, Max, MEF2C, CHOP, MAPKAPK2 (MAPK-activated protein kinase-2), and PRAK kinase (p38-related/ activated protein kinase). p38 MAP kinase is expressed broadly in normal tissues and various cell lines. There are three alternative spliced forms of p38 (CSBP2/p38α, CSBP1, and Mxi2) as well as several homologues including p38β, p38β2, p38γ, and p38δ. These homologues are expressed at different levels in human tissues and can be activated by different, although sometimes overlapping, stress stimuli.
      Principles of the assayThe Calbiochem® PhosphoDetect™ p38 MAP Kinase (Thr¹⁸⁰/Tyr¹⁸²) ELISA Kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for p38 MAP kinase (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing p38 MAP kinase (pThr¹⁸⁰/pTyr¹⁸²), control specimens, and unknowns, are pipetted into these wells. During the first incubation, the p38 MAP kinase antigen binds to the immobilized (capture) antibody. After washing, an antibody specific for p38 MAP kinase phosphorylated at Thr¹⁸⁰/Tyr¹⁸² is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized p38 MAP kinase protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase-labeled anti-rabbit IgG 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 p38 MAP kinase (pThr¹⁸⁰/pTyr¹⁸²) present in the original sample.
      Materials provided• p38 MAP Kinase (pThr¹⁸⁰/pTyr¹⁸²) Standard (Kit Component No. JA7810-1EA): 2 vials, please refer to vial label for quantity and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA7811-25ML): 1 bottle, 25 ml, contains 15 mM sodium azide
      • p38 MAP Kinase Antibody Coated 96-Well Plate (Kit Component No. JA7812-1EA): 96-well plate supplied as twelve 8-well strips coated with p38 MAP kinase antibody
      • Detection Antibody (Kit Component No. JA7813-11ML): 1 bottle, 11 ml, rabbit anti-p38 MAP kinase (pThr¹⁸⁰/pTyr¹⁸²), contains 15 mM sodium azide
      • Secondary Antibody (Kit Component No. JA7814-125UL): 1 vial, 125 µl, supplied as 100X Anti-Rabbit IgG-Horseradish Peroxidase (HRP) concentrate contains 3.3 mM thymol
      • Secondary Antibody Diluent (Kit Component No. JA7815-25ML): 1 bottle, 25 ml, contains 3.3 mM thymol
      • Wash Buffer Concentrate (Kit Component No. JA7816-100ML): 1 bottle, 100 ml, supplied as 25X
      • TMB Substrate (Kit Component No. JA7817-25ML): 1 bottle, 25 ml, ready-to-use stabilized Tetramethylbenzidine
      • Stop Solution (Kit Component No. JA7818-25ML): 1 bottle, 25 ml, ready-to-use
      • Plate Covers (Kit Component No. JA7819-1EA): 3 adhesive plate sealers
      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
      • Lysis Buffer
      • Deionized or distilled H2O
      • PBS
      • 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 recommendations• 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.
      • When not in use, kit components should be stored at 4°C. All reagents should be warmed to room temperature before use.
      • 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.
      • Samples should be frozen if not analyzed shortly after collection, avoiding multiple freeze-thaw cycles. Thaw completely and mix well prior to analysis. If particulate matter is present, centrifuge or filter prior to analysis.
      • All standards, controls, and samples should be run in duplicate.
      • Samples containing p38 MAP kinase (pThr180/pTyr182) 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.
      • When pipetting reagents, maintain a consistent order of addition from well to well. This ensures equal incubation times for all wells.
      • Cover or cap all reagents when not in use.
      • Do not mix or interchange different reagent lots from various kit lots.
      • Read absorbances within 2 h of assay completion.
      • In-house controls should be run with every assay. If control values fall outside pre-established ranges, the accuracy of the assay is questionable.
      • All residual Wash Buffer 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.
      • Because TMB is light sensitive, avoid prolonged exposure to light. Avoid contact between TMB and metal, or color may develop.
      • 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.
      • Guidelines for washing: Incomplete washing will adversely affect the test outcome. All washing must be performed with the Wash Buffer provided. 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. After aspiration, fill the wells with at least 0.4 ml of Diluted Wash Solution. Let soak for 15 to 30 s, then aspirate the liquid. Repeat as directed in the assay procedure that follows. After the washing procedure, invert the plate and tap dry on absorbent towels. If a squirt bottle is used, flood the plate with Wash Buffer, completely filling all wells. After washing, invert the plate and tap dry on absorbent towels. 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.
      Preparation• Cell Lysis Buffer: • 10 mM Tris-HCl, 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 set III (Cat. No. 539134) This buffer is stable for 2-3 weeks at 4°C or for up to 6 months when aliquoted (without protease inhibitors and PMSF added) and stored at -20°C. When stored frozen, the Cell Lysis 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. • Preparation of Cell Lysate: 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 extraction buffer depends on the number of cells in the cell pellet and the expression of p38 MAP kinase (pThr180/pTyr182). For example, 108 Jurkat cells grown in RPMI plus 10% FBS and treated with 100 µM anisomycin can be lysed in 1 ml Cell Lysis Buffer. Under these conditions, the use of 0.1-1 µl of the clarified cell extract diluted to a volume of 100 µl per well in Standard Diluent Buffer is sufficient for the detection of p38 MAP kinase (pThr180/pTyr182). 5. Transfer lysate to microcentrifuge tubes and centrifuge at 13,000 rpm for 10 min at 4°C. 6. Aliquot the clear lysate 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• p38 MAP Kinase (pTpY180/182) Standard Note: The p38 MAP kinase (pThr180/pTyr182) standard is prepared using purified, full length human recombinant p38 MAP kinase, which was phosphorylated by MKK6. One unit of standard is equivalent to the amount of p38 MAP kinase (pThr180/pTyr182) derived from about 40 pg of p38 MAP Kinase phosphorylated by MKK6. 1. Reconstitute p38 MAP kinase (pThr180/pTyr182) standard with Standard Diluent Buffer. Refer to standard vial label for instructions. Swirl or mix gently and allow to sit for 10 min to ensure complete reconstitution. Label as 100 units/ml p38 MAP kinase (pThr180/pTyr182). 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 p38 MAP kinase (pThr180/pTyr182). 3. Make serial dilutions of the standard as described in the following table. Mix thoroughly between steps. 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.

      Table 1: p38 MAP Kinase (pThr180/pTyr182) Standard Dilutions

      • Secondary Antibody: Note: The Secondary Antibody is a viscous solution in 50% glycerol. To ensure accurate dilution, allow it to reach room temperature. Mix gently and pipette slowly. Remove excess solution from pipette tip by gently wiping with clean absorbent paper. Dilute 10 µl Secondary Antibody with 1 ml of Secondary Antibody Diluent for each 8-well strip used in the assay. Label as Anti-Rabbit IgG-HRP Working Solution. To determine the total volume needed, use the table below as a guideline. Return the unused Secondary Antibody to the refrigerator.

      Table 2: Guidelines for Diluting Anti-Rabbit IgG Horseradish Peroxidase (HRP)

      • Wash Buffer: Allow the Wash Buffer Concentrate to reach room temperature. Mix to ensure that any precipitated salts have redissolved. To prepare the Diluted Wash Buffer, mix 1 volume Wash Buffer Concentrate with 24 volumes deionized H2O (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 Diluted Wash Buffer Concentrate and the Wash Buffer in the refrigerator. The Diluted 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. Return the unused strips and frame to the foil pouch and store at 4°C.
      2. Add 100 µl Standard Diluent Buffer to zero wells. Well(s) reserved for chromogen blank should be left empty.
      3. Add 100 µl standards, samples, or controls to the designated 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). Tap gently on side of plate to thoroughly mix. Note: The Cell Lysis Buffer and final dilution chosen should be optimized for each experimental system.
      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 the liquid from the wells and discard. Wash wells 4 times as described in the Guidelines for washing.
      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 the liquid from the wells and discard. Wash the wells 4 times as described in the Guidelines for washing.
      9. Add 100 µl Anti-Rabbit IgG-HRP 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 the liquid from wells and discard. Wash the wells 4 times as described in the Guidelines for washing.
      12. Add 100 µl TMB Substrate 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 TMB Substrate 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 an absorbance of 2.0 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 of 100 µl each TMB Substrate and Stop Solution. Read the plate within 2 h after adding the Stop Solution.
      16. Plot on graph paper 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 p38 MAP kinase(pThr180/pTyr182) 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 curve

      Table 3: Example Standard Values

      Example data obtained using the diluted standards over the range of 0 to 100 units/ml p38 MAP kinase (pThr180/pTyr182).
      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. Samples that contain >100 units/ml should be diluted with Standard Diluent Buffer, re-analyzed, and the results should be multiplied by the appropriate dilution factor. The influence of various lysis buffers has not been thoroughly investigated. The rate of degradation of native p38 MAP kinase or dephosphorylation of p38 MAP kinase (pThr¹⁸⁰/pTyr¹⁸²) in various matrices has not been investigated. Although p38 MAP kinase degradation or dephosphorylation of p38 MAP kinase (pThr¹⁸⁰/pTyr¹⁸²) in the Cell Lysis Buffer used in this User Protocol has not been observed, the possibility cannot be excluded.
      Sensitivity≤0.8 U/ml
      Sensitivity Notesinline-image:FIG>
      Assay Range1.6-100 U/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known p38 MAP kinase (pThr180/pTyr182) concentration were assayed in replicates of 16 to determine precision within an assay.


      Table 5: Inter-Assay Precision

      Samples were assayed 48 times in multiple assays to determine precision between assays.
      RecoveryTo evaluate recovery, unstimulated Jurkat cell lysates were prepared and adjusted to 200 µg/ml total protein. Recombinant p38 MAP kinase (pThr¹⁸⁰/pTyr¹⁸²) was spiked at 3 different concentrations into the cell lysate. The percent recovery was calculated as an average of 97%.
      Parallelism

      Figure 1: Sensitivity

      The analytical sensitivity of this assay is less than 0.8 units/ml human p38 MAP kinase (pThr180/pTyr182). 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 p38 MAP kinase (pThr180/pTyr182) (above) shows that the sensitivity of the ELISA is ~10X greater than that of immunoblotting. The bands shown in the immunoblot were developed using PhosphoDetect™ Anti- p38 MAP kinase (pThr180/pTyr182), Rabbit pAb (Cat. No. 506119) and chemiluminescent detection.
      Linearity

      Table 6: Linearity of Dilution

      Cell Lysis Buffer was spiked with p38 MAP kinase (pThr180/pTyr182) and serially diluted in Standard Diluent Buffer over the range of the assay. Linear regression analysis of sample values versus the expected concentration yielded a correlation coefficient of 0.99.
      Specificity

      Figure 2: p38 MAPK (pThr180/pTyr182) Concentration (Units/ml)

      Natural p38 MAP kinase (pThr180/pTyr182) from anisomycin-treated cells was serially diluted in Standard Diluent Buffer following lysis of the cells. The absorbance of each dilution was plotted against the p38 MAP kinase (pThr180/pTyr182) standard curve. Parallelism demonstrated that the standard accurately reflects full-length p38 MAP kinase (pThr180/pTyr182) content in samples.


      Figure 3: Assay Specificity

      Recombinant p38 MAP kinase was phosphorylated in vitro using MKK6 enzyme. Non-phosphorylated p38 MAP kinase was used as control. The phosphorylated and non-phosphorylated p38 MAP kinase were analyzed as outlined in the Detailed Protocol. As shown above this kit is specific for measurement of phosphorylated p38 MAP kinase at Thr180/Tyr182. The kit does not detect non-phosphorylated p38 MAP kinase protein.


      Figure 4: Peptide Blocking

      The specificity of the assay for p38 MAP kinase phosphorylated at Thr180/Tyr182 was confirmed by peptide competition. The data presented in the figure above shows that only the phosphopeptide containing the phosphorylated threonine and tyrosine could block the ELISA signal. The same sequence containing non-phosphorylated threonine and tyrosine at position 180/182 did not block the signal.
      Registered TrademarksCalbiochem™ is a registered trademark of EMD Chemicals, Inc.
      Triton™ is a registered trademark of Dow Chemical Company
      PhosphoDetect™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.