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CBA032 ERK 1/2 ELISA Kit

ERK 1/2 ELISA Kit: Ficha de datos de seguridad (MSDS o SDS), certificado de análisis y de calidad (CoA y CoQ), expedientes, folletos y otros documentos disponibles.
Detection Methods: Colorimetric
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      Descripción

      Replacement Information

      Tabla espec. clave

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      This product has been discontinued.



      We apologize for the inconvenience, but we do not currently have an alternative product.






      Detects and quantifies the level of ERK 1/2 proteins independent of their phosphorylation state. The ERKs (Extracellular Signal Regulated Kinases) are members of Mitogen Activated Protein Kinases (MAPK) family of proteins that are activated by a variety of mitogenic stimuli as well as differentiation signals. The ERK 1/2 signaling cascade has been shown to be a critical regulator of cell differentiation, cell physiology and neuronal function. Although, this kit is designed for use with human cell lines, platelets, and lymphocytes, it cross-reacts with mouse and rat cells.
      Catalogue NumberCBA032
      Brand Family Calbiochem®
      Application Data
      The sensitivity of this ELISA was compared to immunoblotting using known quantities of ERK1/2. The data presented in Figure 4 show that the sensitivity of the ELISA is ~4x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using an anti ERK1/2 antibody and chemiluminescent detection.
      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 manifold multi channel pipette is desirable for large assays.)
      • Cell Lysis Buffer (see Recommended Formulation in Reagent Preparation).
      • Deionized or distilled H2O.
      • Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.).
      • Graph paper: linear (Cartesian), log log, or semi log, as desired.
      • 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. 1999. Prog. Biophys. Mol. Biol. 71, 479.
      Impey, S., et al. 1999. Neuron 23, 11.
      Tan, P.B., and S.K. Kim, 1999. Trends Genet. 15, 145.
      Xu, S., et al. 1997. Mol. Endocrinol. 11, 1618.
      Cobb, M.H. and E.J. Goldsmith 1995. J. Biol. Chem. 270, 14843.
      Cobb, M.H., et al. 1994. Cancer Biol. 5, 261.
      Payne, D.M., et al. 1991. EMBO J. 10, 885.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, ERK 1/2 Standard, Diluents, Detector Antibody, Secondary Antibody, Wash Buffer, Sample Treatment Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Assay range31.2-2000 pg/ml
      Assay time4 h
      Sample TypeCells
      Physicochemical Information
      Sensitivity< 16 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® ERK1/2 ELISA Kit is designed to detect and quantify the level of ERK1/2 proteins independent of its phosphorylation status. Although performance characterization of the ELISA kit is done primarily on human cell lines, this ELISA kit can be used for detection ERK1/2 in mouse and rat cells. This assay is intended for detection of ERK1/2 from cell lysates and can be used for normalization of ERK1/2 content of the samples when examining quantities of phosphorylated sites on ERK1/2 using another The PhosphoDetect™ ERK1/2 (pThr¹⁸⁵/pTyr¹⁸⁷) ELISA Kit (Cat. No. CBA006).
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, ERK 1/2 Standard, Diluents, Detector Antibody, Secondary Antibody, Wash Buffer, Sample Treatment Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Número de referencia GTIN
      CBA032 0

      Documentation

      ERK 1/2 ELISA Kit Certificados de análisis

      CargoNúmero de lote
      CBA032

      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. 1999. Prog. Biophys. Mol. Biol. 71, 479.
      Impey, S., et al. 1999. Neuron 23, 11.
      Tan, P.B., and S.K. Kim, 1999. Trends Genet. 15, 145.
      Xu, S., et al. 1997. Mol. Endocrinol. 11, 1618.
      Cobb, M.H. and E.J. Goldsmith 1995. J. Biol. Chem. 270, 14843.
      Cobb, M.H., et al. 1994. Cancer Biol. 5, 261.
      Payne, D.M., et al. 1991. EMBO J. 10, 885.

      Folleto

      Cargo
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protein Kinase Assay and Detection Kits Brochure
      Protocolo de usuario

      Revision21-October-2008 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, mouse, rat
      Intended useThe Calbiochem® ERK1/2 ELISA Kit is designed to detect and quantify the level of ERK1/2 proteins independent of its phosphorylation status. Although performance characterization of the ELISA kit is done primarily on human cell lines, this ELISA kit can be used for detection ERK1/2 in mouse and rat cells. This assay is intended for detection of ERK1/2 from cell lysates and can be used for normalization of ERK1/2 content of the samples when examining quantities of phosphorylated sites on ERK1/2 using another The PhosphoDetect™ ERK1/2 (pThr¹⁸⁵/pTyr¹⁸⁷) ELISA Kit (Cat. No. CBA006).
      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 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 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 (within 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 Thr202 and Tyr204 of human ERK1, and on Thr185 and Tyr187 of human ERK2 and the dual phosphorylation is required for 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 transcription factors, such as ELK 1.
      Principles of the assayThe Calbiochem® ERK1/2 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, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the ERK1/2 antigens bind to the immobilized (capture) antibody. After washing, an antibody specific for both ERK1 and ERK2 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 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 ERK1/2 present in the original specimen.
      Materials provided• ERK1/2 Standard (Kit Component No. JA8043-1EA): 2 vials, refer to vial label for quantity and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA8044-25ML): 1 bottle, 25 ml, contains 15 mM sodium azide
      • Sample Treatment Buffer (Kit Component No. JA9370-10ML): 1 bottle, 10 ml
      • ERK1/2 Antibody Coated Wells (Kit Component No. JA8045-1EA): 1 plate, 96-wells
      • Rabbit anti ERK1/2 (Detection Antibody) (Kit Component No. JA8046-11ML): 1 bottle, 11 ml, contains 15 mM sodium azide
      • Anti rabbit IgG Horseradish Peroxidase (HRP) Concentrate (Kit Component No. JA8047-125UL): 1 vial, 0.125 ml, supplied as 100X, contains 3.3 mM thymol
      • HRP Diluent (Kit Component No. JA8048-25ML): 1 bottle, 25 ml, contains 3.3 mM thymol
      • Wash Buffer Concentrate (Kit Component No. JA8049-100ML): 1 bottle, 100 ml, supplied as 25X
      • Soluble Substrate (Kit Component No. JA8050-25ML): 1 bottle, 25 ml, Tetramethylbenzidine (TMB)
      • Stop Solution (Kit Component No. JA8051-25ML): 1 bottle, 25 ml
      • Plate Sealers (Kit Component No. JA8052-1EA): 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 manifold multi channel pipette is desirable for large assays.)
      • Cell Lysis Buffer (see Recommended Formulation in Reagent Preparation).
      • Deionized or distilled H2O.
      • Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.).
      • Graph paper: linear (Cartesian), log log, or semi log, as desired.
      • Glass or plastic tubes for diluting and aliquoting standard.
      • Absorbent paper towels.
      • Calibrated beakers and graduated cylinders in various sizes.
      Precautions and recommendations• Disposal Note: This kit contains materials with small quantities of sodium azide. 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 refrigerated. All reagents should be warmed to room temperature before use.
      • Plates should be allowed to come to room temperature before opening the foil bag. 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. Avoid multiple freeze thaw cycles of samples. Thaw completely and mix well prior to analysis.
      • If large amounts of particulate matter are present, centrifuge or filter prior to analysis.
      • It is recommended that all standards, controls and samples be run in duplicate.
      • Extracted cell lysate samples containing ERK1/2 proteins should be diluted with Standard Diluent Buffer at least 1:10. This dilution is necessary to reduce the matrix effect of the cell lysate 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 suspect.
      • 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.
      • Because Soluble Substrate is light sensitive, avoid prolonged exposure to light. Also avoid contact between Soluble Substrate and metal, or color may develop.
      • Safety: All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing infectious agents.
      • Directions for Washing: Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided. Washing can be performed manually as follows: completely aspirate the liquid from all wells by gently lowering an aspiration tip (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 Detailed Protocol. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, completely filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. If using an automated washer, the operating instructions for washing equipment should be carefully followed. If your automated washer allows, 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% to 1.0% SDS • 0.5% deoxycholate • Protease Inhibitor Cocktail Set III (e.g. 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 before using. The stability of protease inhibitor supplemented Cell Extraction Buffer is 24 h at 4°C. • Preparation of Cell Lysate: This protocol has been applied to several cell lines with the Cell Extraction Buffer above. Researchers may optimize the cell extraction procedures that work best in their hands. 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 Extraction Buffer depends on the cell number in cell pellet and expression of ERK1/2. For example, 1 x 108 Jurkat cells grown in RPMI plus 10% FBS can be extracted in 1 ml of Extraction Buffer. Under these conditions, use of 0.1 1 µl of the clarified cell extract diluted to a volume of 100 µl/well in Standard Diluent Buffer (See Detailed Protocol) is sufficient for the detection of ERK1/2. 5. Transfer extracts 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. • Sample Treatment: This ELISA kit efficiently detects ERK1/2 proteins in denatured cell extracts. The following sample pretreatment options are intended to maximize ERK protein availability for measurement. Sample treatment is necessary if cells are lysed using the Cell Lysis Buffer recommended above. The 3 sample treatment options include addition of Sample Treatment Buffer (included), 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 with Standard Diluent Bufffer. For example, for duplicate analyses, add 20 µl sample and 20 µl Sample Treatment Buffer, then after incubation, ad 160 µl Standard Diluent Buffer. The dilution chosen should be optimized for each experimental system. 2. SDS treatment: Cell lysates containing 2.5 10 mg/ml of total protein should 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 microplate. This means that the extracts should be diluted 1:25 to 1:100 in Standard Diluent Buffer to obtain a final SDS concentration of ~0.01%. In the following figure, extracts containing 10, 5, 2.5 and 1 mg/ml of total protein from Jurkat cells were treated by 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 show that cell lysates with the addition of 0.25 1% SDS generate higher signals than the extract with 0.1% SDS alone.

      Figure 1: Effect of SDS Concentration

      3. Boiling the samples: Cell lysates containing <2.5 mg/ml of total protein should be extracted with the Cell Lysis Buffer containing 0.1% SDS, then heated in boiling water for 5 min, cooled and centrifuged before loading in the ERK ELISA microplate. In the following figure, cell extracts containing total protein concentrations of 8, 5, 2 and 1 mg/ml were boiled for 5 min, centrifuged and assayed (10 µg). The data show that protein concentration affects the signal of this ELISA, perhaps due to protein aggregation or precipitation after boiling. We recommend heating samples in Cell Lysis Buffer (with 0.1% SDS) when samples are <2.5 mg/ml protein. If samples are >2.5 mg/ml, an elevated concentration of SDS in the cell extract is recommended.

      Figure 2: Effect of Protein Concentration

      Performance Comparison of the Various Sample Treatment Methods and Cell Lysis Buffers: Jurkat cells were treated with 100 ng/ml PMA for 10 min and cell lysates were prepared. In the following figure, cell lysate prepared with Cell Extraction Buffer either boiled for 5 min, treated with Sample Treatment Buffer, or left untreated. Cell lysate was also prepared using Denaturing Cell Lysis Buffer. Samples were then analyzed using the ERK1/2 and PhosphoDetect™ ERK ERK1/2 (pThr185/pTyr187) ELISA Kits.

      Figure 3: Sample Treatment Comparisons
      Reagent preparation• Reconstitution and Dilution of ERK1/2 Standard: Note: This ERK1/2 standard was prepared from purified, full length, recombinant human ERK2 protein expressed in E. coli. 1. Reconstitute ERK1/2 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 2000 pg/ml ERK1/2. Use standard within 1 h of reconstitution. 2. Add 0.25 ml of Standard Diluent Buffer to each of 6 tubes labeled 1000, 500, 250, 125, 62.5, and 31.2 pg/ml ERK1/2. 3. Make serial dilutions of the standard as described in the following dilution 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: Dilution of ERK1/2 Standard

      • Storage and Final Dilution of Anti rabbit IgG Horseradish Peroxidase (HRP): Please Note: The Anti rabbit IgG HRP 100X concentrate is in 50% glycerol. This solution is viscous. To ensure accurate dilution, allow 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. 1. Dilute 10 µl of this 100x concentrated solution with 1 ml of HRP Diluent for each 8 well strip used in the assay. Label as Anti rabbit IgG HRP Working Solution.

      Table 2: Example Anti-rabbit IgG-HRP Working Solution

      2. Return the unused Anti rabbit IgG HRP concentrate to the refrigerator. • Dilution of Wash Buffer: Allow the 25x 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 (e.g., 50 ml may be diluted up to 1.25 liters, 100 ml may be diluted up to 2.5 liters). Label as Working Wash Buffer. Store both the concentrate and the Working Wash Buffer in the refrigerator. The diluted buffer should be used within 14 days.
      Detailed protocolBe sure to read the Precautions and Recommendations section before carrying out the assay.

      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 current use. (Re bag extra strips and frame. Store these in the refrigerator 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 in Standard Diluent Buffer to maintain a final SDS concentration of 0.01%. The dilution chosen should be optimized for each experimental system. Tap gently on side of plate to thoroughly mix. (See Reagent Preparation, Section B.)
      4. Cover plate with plate sealer and incubate for 2 h at room temperature. Alternatively, the plate may be incubated overnight at 4°C.
      5. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing.
      6. Pipette 100 µl of anti ERK1/2 (Detection Antibody) solution into each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
      7. Cover plate with plate sealer and incubate for 1 h at room temperature.
      8. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing.
      9. Add 100 µl anti rabbit IgG HRP Working Solution to each well except the chromogen blank(s). (Prepare the working dilution as described in Reagent Preparation, Section C.)
      10. Cover plate with the plate sealer and incubate for 30 min at room temperature.
      11. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing.
      12. Add 100 µl of Soluble 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. Please Note: Do not cover the plate with aluminum foil or metalized mylar. The incubation time for chromogen substrate is often determined by the plate reader used. Many plate readers have the capacity to record a maximum absorbance 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 only be read 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 of Stop Solution to each well. Tap 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 Soluble 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 ERK1/2 concentrations for unknown samples and controls from the standard curve plotted in step 16. Multiply value(s) obtained for sample(s) by dilution factor to correct for the dilution in step 3. (Samples still producing signals higher than the highest standard (2000 pg/ml) should be further diluted in Standard Diluent Buffer and re analyzed, multiplying the concentration found by the appropriate dilution factor.)
      Standard curve

      Table 3: Typical Data Obtained from Diluted Standards

      The above data was obtained for the various standards over the range of 0 to 2000 pg/ml ERK1/2.
      Limitations of the assayDo not extrapolate the standard curve beyond the 2000 pg/ml standard point; the dose response is non linear in this region and accuracy is difficult to obtain. Dilute samples >2000 pg/ml with Standard Diluent Buffer; re analyze these and multiply results by the appropriate dilution factor.
      The influence of various extraction buffers has not been thoroughly investigated. The rate of degradation of native ERK1/2 in various matrices has not been investigated.
      Sensitivity< 16 pg/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <16 pg/ml of human total ERK1/2. 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. The data presented in Figure 4 show that the sensitivity of the ELISA is ~4x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using an anti ERK1/2 antibody and chemiluminescent detection.
      Assay Range31.2-2000 pg/ml
      Precision

      Table 4: Intra-Assay Precision

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


      Table 5: Inter-Assay Precision

      Samples were assayed 36 times in multiple assays to determine precision between assays.
      RecoveryTo evaluate recovery, ERK1/2 standard was spiked at 3 different concentrations into 10% cell extract buffer. The percent recovery was calculated as an average of 95%.
      Parallelism

      Figure 5: Parallelism

      Natural ERK1/2 from human Jurkat cell lysates were serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the ERK1/2 standard curve. Parallelism was demonstrated by the figure below and indicates that the standard accurately reflects ERK1/2 content in samples.
      Linearity

      Table 6: Linearity of Dilution

      Jurkat cells were grown in tissue culture medium containing 10% fetal calf serum 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 content. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99 in both cases.
      SpecificityThe ERK1/2 ELISA kit is specific for measurement of ERK1/2 proteins. It shows no cross reactivity with JNK (c Jun NH2 terminal kinase) and p38 MAPK.

      Figure 6: ERK ELISAs on Jurkat Cells

      The detection of ERK1/2 is independent of phosphorylation status. As shown in Figure 6, Jurkat cells were treated with 50 ng/ml PMA for 10 min and untreated Jurkat cells were used as control. The cell lysates were analyzed with ERK1/2 ELISA and PhosphoDetect™ ERK1/2 (pThr185/pTyr187) ELISA kit (Cat. No. CBA006). This ERK1/2 ELISA kit detected both phosphorylated and non phosphorylated ERK1/2 in PMA treated cells and untreated control. Whereas the ERK1/2 (pThr185/pTyr187) ELISA kit detected the phosphorylated ERK1/2 in PMA treated Jurkat cells, not the non phosphorylated ERK1/2 in untreated Jurkat cells.


      Figure 7: Sensitivity

      Figure 7 shows that ERK1/2 ELISA is useful to normalize the ERK1/2 content in cells when an ERK1/2 (pThr185/pTyr187) phospho site specific ELISA is used. 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 (pThr185/pTyr187). The amount of ERK1/2 remains constant, while the level of phosphorylation at Thr185 and Tyr187 increases with the dosage of PMA. The results correlated very well with the results from immunoblotting (inset in the graph above).
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