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CBA006 PhosphoDetect™ ERK1/2 (pThr¹⁸⁵/pTyr¹⁸⁷) ELISA Kit

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Sinónimos: p44 MAP Kinase, Phospho-Specific (Thr¹⁸⁵/Tyr¹⁸⁷) ELISA

Detection Methods: Colorimetric
CBA006
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Tabla espec. clave

Species ReactivityDetection Methods
H, M, RColorimetric

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      Description
      Overview

      This product has been discontinued.



      Detects the dual phosphorylated forms of ERK1 at Thr202 and Tyr204 and ERK2 at Thr185 and Tyr187. This activation occurs as a result of treatment with a large variety of stimuli including mitogens, cytokines, and growth factors. Although this kit was designed for human samples, it cross-reacts with mouse and rat.
      Catalogue NumberCBA006
      Brand Family Calbiochem®
      Synonymsp44 MAP Kinase, Phospho-Specific (Thr¹⁸⁵/Tyr¹⁸⁷) ELISA
      Application Data
      Jurkat cells were treated with 100 ng/ml PMA for 10 min and cell lysates were prepared. Cell lysate prepared with Cell Lysis Buffer was either boiled for 5 min, treated with Sample Treatment Buffer, or not treated. Cell lysate was also prepared using Denaturing Cell Lysis Buffer. Samples were analyzed with the ERK1/2 and the PhosphoDetect™ ERK (Thr185/Tyr187) ELISA Kits.

      The sensitivity of this ELISA was compared to immunoblotting using known quantities of ERK1/2 Phospho-Thr185/Tyr187. The data shows that the sensitivity of the ELISA is approximately 4X greater than that of immunoblotting. The bands shown in the immunoblotting were developed using rabbit anti-ERK1/2 Phospho-Thr185/Tyr187 and an alkaline phosphatase conjugated anti-rabbit IgG followed by chemiluminescent detection.

      Natural ERK1/2 Phospho-Thr185/Tyr187 from PMA-treated Jurkat cell lysates was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the ERK1/2 Phospho-Thr185/Tyr187 standard curve. Parallelism is demonstrated in the figure above and indicates that the standard accurately reflects natural ERK1/2 Phospho-Thr185/Tyr187 content in samples.
      Materials Required but Not Delivered• Plate reader capable of measurement at or near 450 nm
      • Calibrated adjustable precision pipettes, preferably with disposable plastic tips; a multi-channel pipette is desirable for large assays
      • Cell lysis buffer (see recommended formulation below)
      • Deionized or distilled H2O
      • Automated or manual plate washer such as a squirt bottle or a 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
      ReferencesJanulis, M., et al. 2001. Mol. Cell. Biol. 21, 2235.
      Nanki, T., et al. 2001. J. Immunol. 167, 5381.
      Sweatt, J.D. 2001. J. Neurochem. 76, 1.
      Cross, T.G., et al. 2000. Exp. Cell Res. 256, 34.
      Kolch, W. 2000. Biochem. J. 351, 289.
      McCubrey, J.A., et al. 2000. Leukemia 14, 9.
      Pearson, G., et al. 2000. J. Biol. Chem. 275, 37303.
      Cobb, M.H., et al. 1994. Semin. Cancer Biol. 5, 261.
      Cobb, M.H. and E.J. Goldsmith. 1995. J. Biol. Chem. 270, 14843.
      Cobb, M.H. 1999. Prog. Biophys. Mol. Biol. 71, 479.
      Impey, S., et al. 1999. Neuron 23, 11.
      Payne, D.M., et al. 1991. EMBO J. 10, 885.
      Tan, P.B., and S.K. Kim. 1999. Trends Genet. 15, 145.
      Xu, S., et al. 1997. Mol. Endocrinol. 11, 1618.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ Standard, Diluents, Detector Antibody, Secondary Antibody, Sample Treatment Buffer, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Applications
      Biological Information
      Assay range1.6-100 units/ml
      Assay time4 h
      Sample TypeCell lysates
      Species Reactivity
      • Human
      • Mouse
      • Rat
      Physicochemical Information
      Sensitivity≤0.8 units/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: 26-36/37-45

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      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
      Intended useThe Calbiochem® ERK1/2, Phospho-Specific (Thr¹⁸⁵/Tyr¹⁸⁷) ELISA is designed to detect and quantify the level of both dual-phosphorylated ERK2 at threonine 185 and tyrosine 187 (ERK2, Phospho-Specific Thr¹⁸⁵/Tyr¹⁸⁷) and ERK1 at threonine 202 and tyrosine 204 (ERK1, Phospho-Specific Thr²⁰²/Tyr²⁰⁴). The level of the phosphorylation of ERK1/2 can be an indirect indication of the activity of upstream kinases of ERK1/2 or the activity of ERK1/2 themselves. Although performance characterization of this ELISA kit is done primarily on human cell lines, this ELISA kit can be used for the detection of ERK1/2 in mouse and rat cells. This assay is intended for the detection of ERK1/2 dual phosphorylation from cell lysates.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arricval 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, ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ Standard, Diluents, Detector Antibody, Secondary Antibody, Sample Treatment Buffer, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Specifications
      Global Trade Item Number
      Número de referencia GTIN
      CBA006-1KIT 04055977220834

      Documentation

      PhosphoDetect™ ERK1/2 (pThr¹⁸⁵/pTyr¹⁸⁷) ELISA Kit Certificados de análisis

      CargoNúmero de lote
      CBA006

      Referencias bibliográficas

      Visión general referencias
      Janulis, M., et al. 2001. Mol. Cell. Biol. 21, 2235.
      Nanki, T., et al. 2001. J. Immunol. 167, 5381.
      Sweatt, J.D. 2001. J. Neurochem. 76, 1.
      Cross, T.G., et al. 2000. Exp. Cell Res. 256, 34.
      Kolch, W. 2000. Biochem. J. 351, 289.
      McCubrey, J.A., et al. 2000. Leukemia 14, 9.
      Pearson, G., et al. 2000. J. Biol. Chem. 275, 37303.
      Cobb, M.H., et al. 1994. Semin. Cancer Biol. 5, 261.
      Cobb, M.H. and E.J. Goldsmith. 1995. J. Biol. Chem. 270, 14843.
      Cobb, M.H. 1999. Prog. Biophys. Mol. Biol. 71, 479.
      Impey, S., et al. 1999. Neuron 23, 11.
      Payne, D.M., et al. 1991. EMBO J. 10, 885.
      Tan, P.B., and S.K. Kim. 1999. Trends Genet. 15, 145.
      Xu, S., et al. 1997. Mol. Endocrinol. 11, 1618.

      Folleto

      Cargo
      Protein Kinase Assay and Detection Kits Brochure
      Protocolo de usuario

      Revision11-June-2010 JSW
      Synonymsp44 MAP Kinase, Phospho-Specific (Thr¹⁸⁵/Tyr¹⁸⁷) ELISA
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, mouse, rat
      StorageUpon arricval store the entire contents of the kit at 4°C.
      Intended useThe Calbiochem® ERK1/2, Phospho-Specific (Thr¹⁸⁵/Tyr¹⁸⁷) ELISA is designed to detect and quantify the level of both dual-phosphorylated ERK2 at threonine 185 and tyrosine 187 (ERK2, Phospho-Specific Thr¹⁸⁵/Tyr¹⁸⁷) and ERK1 at threonine 202 and tyrosine 204 (ERK1, Phospho-Specific Thr²⁰²/Tyr²⁰⁴). The level of the phosphorylation of ERK1/2 can be an indirect indication of the activity of upstream kinases of ERK1/2 or the activity of ERK1/2 themselves. Although performance characterization of this ELISA kit is done primarily on human cell lines, this ELISA kit can be used for the detection of ERK1/2 in mouse and rat cells. This assay is intended for the detection of ERK1/2 dual phosphorylation from cell lysates.
      BackgroundERK (Extracellular Signal Regulated Kinase), also known as MAPK (Mitogen-Activated Protein Kinase), has two closely related isoforms. ERK1, also known as MAP kinase 1 or p44 MAP kinase, is a 44 kDa protein and ERK2, also known as MAP kinase 2 or p42 MAP kinase, is a 42 kDa protein. These kinases belong to a family of serine/threonine kinases that are activated upon treatment of the cells with a large variety of stimuli including mitogens, hormones, growth factors, cytokines, and bioactive peptides. Cell stimulation induces the activation of a signaling cascade, the downstream effects of which have been linked to the regulation of cell growth and differentiation as well as the regulation of the cytoskeleton. ERK1 and ERK2 are serine/threonine kinases expressed broadly in normal tissues and various cell lines. They are activated through the phosphorylation of a threonine and a tyrosine residue (in a Thr-Glu-Tyr motif) within the activation loop by MEKs (MAPK/ERK kinases), including MEK1 (MAPK/ERK kinase 1) or MEK2. The phosphorylation occurs on threonine 202 and tyrosine 204 of human ERK1, and on threonine 185 and tyrosine 187 of human ERK2, and dual phosphorylation is required for the enzyme activity of ERK1 and ERK2. Once activated, ERK1 and ERK2 can phosphorylate PXS/TP motifs in many different proteins including cytoskeletal proteins, translation regulators, the Rsk family of protein kinases and transcriptionfactors, such as ELK-1.
      Principles of the assayThe The Calbiochem® ERK1/2, PhosphoDetect™ (Thr¹⁸⁵/Tyr¹⁸⁷) ELISA Kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for ERK1/2 (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷, control specimens, and unknowns are pipetted into the wells. During the first incubation, the ERK1/2 antigens bind to the immobilized (capture) antibody. After washing, an antibody specific for ERK1/2 phosphorylated at Thr¹⁸⁵ and Tyr¹⁸⁷ is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized ERK1/2 proteins 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-part sandwich. After a third incubation and washing to remove 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 ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ present in the original specimen.
      Materials provided• ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ Standard (Kit Component No. JA7790-1EA): 2 vials, lyophilized standard, please refer to vial label for formulation and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA7791-25ML): 1 bottle, 25 ml buffer containing 15 mM sodium azide
      • ERK Antibody Coated 96-Well Plate (Kit Component No. JA7792-1EA): 1 plate, 96-wells, coated with ERK1/2 antibody
      • Detector Antibody (Kit Component No. JA7793-11ML): 1 vial, 11 ml rabbit anti-ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ antibody containing 15 mM sodium azide
      • Secondary Antibody (Kit Component No. JA7794-125UL): 1 vial, 125 µl anti-rabbit IgG-Horseradish Peroxidase (HRP) Concentrate, supplied as 100X in 50% glycerol containing 3.3 mM thymol
      • Secondary Antibody Diluent (Kit Component No. JA7795-25ML): 1 bottle, 25 ml containing 3.3 mM thymol
      • Sample Treatment Buffer (Kit Component No. JA9371-10ML): 1 vial, 10 ml
      • Wash Buffer Concentrate (Kit Component No. JA7796-100ML): 1 bottle, 100 ml, supplied at 25X
      • TMB Substrate (Kit Component No. JA7797-25ML): 1 bottle, 25 ml Tetramethylbenzidine
      • Stop Solution (Kit Component No. JA7798-25ML): 1 bottle, 25 ml, ready-to-use
      • Plate Covers (Kit Component No. JA7799-1EA): 3 adhesive strips
      Materials Required but not provided• Plate reader capable of measurement at or near 450 nm
      • Calibrated adjustable precision pipettes, preferably with disposable plastic tips; a multi-channel pipette is desirable for large assays
      • Cell lysis buffer (see recommended formulation below)
      • Deionized or distilled H2O
      • Automated or manual plate washer such as a squirt bottle or a 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 refrigerated. 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. Avoid multiple freeze-thaw cycles of frozen samples. Thaw 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 ERK1/2 Phospho-Thr185/Tyr187 protein extracted from cells should be diluted at least 1:10 with Standard Diluent Buffer. This dilution is necessary to reduce the matrix effect of the Cell Lysis Buffer. The SDS concentration resulting from the Cell Lysis Buffer should be less than 0.01% before adding to the plate.
      7. When pipetting reagents, maintain a consistent order of addition from well-to-well to ensure 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 suspect.
      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. Never insert absorbent paper directly into the wells.
      13. Because TMB Substrate is light sensitive, avoid prolonged exposure to light. Avoid contact between TMB Substrate and metal or color may develop.
      14. This kit contains materials with small quantities of sodium azide, which 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.
      15. Washing Procedure: Incomplete washing will adversely affect the test outcome. All washing must be performed with the Wash Buffer supplied with the kit. Completely aspirate the liquid from all wells by gently lowering an aspiration tip or aspiration device 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 under assay procedure. After washing the plate should be inverted and tapped dry on absorbent paper towels. If a squirt bottle is used, flood the plate with wash buffer to completely fill all wells. After washing the plate should be inverted and gently tapped dry on absorbent paper towels. If using an automated washer follow the operating instructions for the washing equipment. 30 s soak cycles should be programmed into the wash cycle.
      PreparationCell 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 Na3O4V • 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. Important: add the protease inhibitors just prior to use. The stability of protease inhibitor supplemented Cell Lysis Buffer is 24 h at 4°C. PMSF is very unstable and must be added prior to use, even if added previously. Note: 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 the protease inhibitors just prior to use. The stability of protease inhibitor supplemented Cell Lysis Buffer is 24 h at 4°C. PMSF is very unstable and must be added just prior to use, even if added previously. • Preparation of cell Lysate Note: This protocol has been successfully applied to several cell lines of human, mouse, or rat origin. Researchers should optimize the cell lysis procedures 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. (At this point the cell pellet can 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, with vortexing at 10-min intervals. The volume of Cell Lysis Buffer depends on the cell number in the cell pellet and the expression of ERK1/2 Phospho-Thr185/Tyr187. For example, 108 Jurkat cells grown in RPMI plus 10% FBS and treated with 50 ng/ml PMA can be extracted in 1 ml Cell Lysis Buffer. Under these conditions, 0.1-1 µl of the clarified cell lysate diluted to a volume of 100 µl/well in Standard Diluent Buffer is sufficient for the detection of ERK1/2 Phospho-Thr185/Tyr187. 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. These samples are ready for assay. Lysates can be stored at -80°C. Avoid multiple freeze/thaw cycles. • Use of Denaturing Cell Lysis Buffer If cells are lysed using a denaturing Cell Lysis Buffer lysates will not have to undergo sample treatment. Dilute sample at least 10-fold with Standard Diluent Buffer prior to running in the ELISA. For example, for a single analysis, add 10 µl sample to 90 µl Standard Diluent Buffer. The dilution chosen should be optimized for each experimental system. See Figure 3 for a performance comparison of the Cell Lysis Buffers. • Sample Treatment This ELISA kit efficiently detects ERK1/2 proteins in denatured cell lysates. The following sample pretreatment options are intended to maximize ERK protein availability for measurement. Sample treatment is necessary if cells are lysed using Cell Lysis Buffer. The three sample treatment options include: addition of Sample Treatment Buffer, SDS treatment, or boiling of samples. The treatment chosen should be optimized for each experimental method. See Figure 3 for a performance comparison of the various sample treatment methods. 1. Sample Treatment Buffer: When cells are lysed with the recommended Cell Lysis Buffer, incubate each sample and control with an equal volume of Sample Treatment Buffer on ice for 20 min. Dilute this mixture at least 5-fold in Standard Diluent Buffer. For example, for duplicate analyses, add 20 µl sample and 20 µl Sample Treatment Buffer, then after incubation, add 160 µl Standard Diluent Buffer. The dilution chosen should be optimized for each experimental system. 2. SDS treatment: Cell lysates may alternatively be treated with the addition of SDS (0.25-1%). Upon extraction, dilution of the lysates in Standard Diluent Buffer must be done prior to loading into the wells of the ERK plate. This means that the lysates should be diluted 1:25 to 1:100 in Standard Dilution Buffer to obtain a final SDS concentration of ~0.01%.

      Figure 1: Effect of SDS Concentration in Cell Lysates on ERK1/2 [pTpY185/187] ELISA Signal

      Lysates containing 10, 5, 2.5 and 1 mg/ml total protein from PMA-treated Jurkat cells were treated by the addition of 1, 0.5, 0.25 and 0.1% of SDS respectively. The cell lysates were then diluted 1:100, 1:50, 1:25, and 1:10 in standard diluent buffer to maintain a final concentration of 0.01% SDS. Upon dilution, equal aliquots of 10 µg total protein were loaded in the ELISA. The data shows that cell extracts with the added of 0.25-1% SDS generate higher signals than the extract with 0.1% SDS alone.

      3. Boiling the samples: Prepare cell lysates using Cell Lysis Buffer. Cell lysates containing >1 mg/ml of total protein should be diluted to ≤1 mg/ml with Cell Lysis Buffer, then heated in boiling water for 5 min, cooled, and centrifuged before diluting with Standard Diluent Buffer and loading in the ERK ELISA plate.

      Figure 2: Effect of Protein Concentration on ERK1/2 [pTpY185/187] Recovery in Boiled Cell

      Cell lysates containing total protein concentrations of 8, 5, 2, and 1 mg/ml were boiled for 5 min, centrifuged, and assayed (10 µg). The data shown show that protein concentration affects the signal of this ELISA, perhaps due to protein aggregation or precipitation after boiling

      Figure 3: ERK1/2 (Total) and ERK1/2 [pTpY185/187] Sample Treatment Comparisons

      Jurkat cells were treated with 100 ng/ml PMA for 10 min and cell lysates were prepared. Cell lysate prepared with Cell Lysis Buffer was either boiled for 5 min, treated with Sample Treatment Buffer, or not treated. Cell lysate was also prepared using Denaturing Cell Lysis Buffer. Samples were analyzed with the ERK1/2 and the PhosphoDetect™ ERK (Thr185/Tyr187) ELISA Kits.
      Reagent preparation• Reconstitution and Dilution of ERK1/2 Phospho-Thr185/Tyr187 Standard The ERK1/2 Phospho-Thr185/Tyr187 Standard was prepared using purified full length human recombinant active ERK2. One unit of standard is equivalent to the amount of ERK1/2 Phospho-Thr185/Tyr187 derived from 40 pg of ERK1/2 that was phosphorylated by MEK1. 1. Reconstitute ERK1/2 Phospho-Thr185/Tyr187 standard with Standard Diluent Buffer according to standard vial label. Swirl or mix gently and allow to sit for 10 min to ensure complete reconstitution. DO NOT VORTEX. Label as 100 units/ml ERK1/2 Phospho-Thr185/Tyr187. 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 ERK1/2 Phospho-Thr185/Tyr187. Mix by gentle inversion. DO NOT VORTEX. 3. Make serial dilutions of the standard as described in the following dilution table. Mix thoroughly between steps. DO NOT VORTEX. 4. The remaining reconstituted standard should be discarded or frozen at -80°C. The standard can be frozen and thawed one time without loss of immunoreactivity.

      Table 1: Dilution of ERK1/2 Phospho-Thr185/Tyr187 Standard

      • Anti-Rabbit IgG Horseradish Peroxidase Please Note: The (Secondary Antibody) is supplied in 50% glycerol, so the solution is viscous. To ensure accurate dilution, allow the Anti-Rabbit IgG-HRP Concentrate to reach room temperature. Gently mix. Pipette Anti-Rabbit IgG-HRP Concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper. Dilute 10 µl of this 100X concentrated solution with 1 ml of HRP diluent for each 8-well strip used. This is the anti-rabbit IgG-HRP working solution; it should be used within 1 h. • Wash Buffer Allow the 25X Wash Buffer Concentrate to reach room temperature and mix to ensure that any precipitated salts have redissolved. Dilute 1 volume of the 25X 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). This is the working wash buffer. Store the concentrate and the working wash buffer in the refrigerator. The diluted buffer should be used within 14 days.
      Detailed protocolAllow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use. Note: 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 current use. (Re-bag extra strips and frame. Store these in the refrigerator for future use.)
      2. Add 100 µl Standard Diluent Buffer to the zero wells. Well(s) reserved for chromogen blank should be left empty.
      3. Add 100 µl standards, samples, or controls to the appropriate wells. Samples prepared in Cell Lysis Buffer must be diluted in Standard Diluent Buffer to maintain a final SDS concentration of 0.01%. The dilution chosen should be optimized for each experimental system. Tap the side of plate gently to mix thoroughly.
      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 solution from the wells and discard the liquid. Wash wells 4 times as described previously.
      6. Pipette 100 µl Detector Antibody to each well except the chromogen blank(s). Tap the side of the plate gently to mix.
      7. Cover the wells with a Plate Cover and incubate for 1 h at room temperature.
      8. Thoroughly aspirate or decant the solution from the wells and discard the liquid. Wash wells 4 times.
      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 solution from the wells and discard the liquid. Wash wells 4 times.
      12. Add 100 µl of TMB Substrate to each well. The liquid in the wells will begin to turn blue.
      13. Incubate for 30 min at room temperature and in the dark. Note: Do not cover the plate with aluminum foil or
      metalized mylar. The incubation time for 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 exceed 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 of 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 composed of 100 µl each of 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. The background absorbance should 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 ERK1/2 Phospho-Thr185/Tyr187 concentrations for the unknown samples and the controls from the standard curve plotted in step 16.
      18. Multiply value(s) obtained for sample(s) 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.
      Assay characteristics and examplesThe following data was obtained for the various standards over the range of 0 to 100 units/ml of ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷.

      Table 2: Intra-Assay Precision

      Samples of known concentration of ERK1/2 Phospho-Thr185/Tyr187 were assayed in replicates of 16 to determine precision within an assay.

      Limitations of the assayDo not extrapolate the standard curve beyond 100 units/ml, as the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute these samples with Standard Diluent Buffer and multiply the results by the appropriate dilution factor.

      While the influence of various lysis buffers has not been thoroughly investigated, ERK1/2 degradation or dephosphorylation of ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ in the Cell Lysis Buffer described in this protocol has not yet been seen.
      Sensitivity≤0.8 units/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <0.8 units/ml of ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷. This was determined by adding two standard deviations to the mean absorbance obtained when the zero standard was assayed 30 times.

      Figure 4: Sensitivity

      The sensitivity of this ELISA was compared to immunoblotting using known quantities of ERK1/2 Phospho-Thr185/Tyr187. The data shows that the sensitivity of the ELISA is approximately 4X greater than that of immunoblotting. The bands shown in the immunoblotting were developed using rabbit anti-ERK1/2 Phospho-Thr185/Tyr187 and an alkaline phosphatase conjugated anti-rabbit IgG followed by chemiluminescent detection.
      Assay Range1.6-100 units/ml
      Precision

      Table 3: Inter-Assay Precision

      Samples were assayed 48 times in multiple assays to determine precision between assays.


      Table 4: Linearity

      Jurkat cells were PMA-treated and lysed with Cell Lysis Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for ERK1/2 Phospho-Thr185/Tyr187 content. Linear regression analysis of sample values versus the expected concentration yielded a correlation coefficient of 0.99.
      RecoveryTo evaluate recovery, ERK1/2 standard was spiked at 3 different concentrations in 10% Cell Lysis Buffer. The percent recovery was calculated as an average of 95%.
      Parallelism

      Figure 5: ERK1/2 (pThr185/pTyr187) ELISA: Parallelism

      Natural ERK1/2 Phospho-Thr185/Tyr187 from PMA-treated Jurkat cell lysates was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the ERK1/2 Phospho-Thr185/Tyr187 standard curve. Parallelism is demonstrated in the figure above and indicates that the standard accurately reflects natural ERK1/2 Phospho-Thr185/Tyr187 content in samples.
      Specificity

      Figure 6: Peptide Competition

      The specificity of this assay for dually phosphorylated ERK1/2 was confirmed by peptide competition. The data shows that only dual phospho-peptide containing the phosphorylated threonine and tyrosine could block the ELISA signal. The same sequence containing non-phosphorylated threonine and tyrosine at position 185/187 or mono-phospho peptides did not block the signal by more then 10%.


      Figure 7: ERK1/2 (Total) and [pTpY185/187] Dose Response of PMA Stimulation on Jurkat Cell

      Jurkat cells were treated with PMA at varying concentrations (0.1 to 1000 ng/ml) for 10 min, lysed and quantitated in parallel for ERK1/2 content (both ERK1/2 and ERK1/2 Phospho-Thr185/Tyr187). The amount of ERK1/2 remains constant, while the levels of phosphorylation at threonine 185 and tyrosine 187 increases with the dosage of PMA. The results correlate well with Immunoblot analysis of the same samples (see inset in the graph above).
      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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