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CBA035 PhosphoDetect™ STAT1 (pTyr⁷⁰¹) ELISA Kit

CBA035
  
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      Overview

      Replacement Information
      Description
      OverviewDetects and quantifies the level of the STAT1 phosphorylated at Tyr701. STAT1 is a member of STAT family of proteins that phosphorylates at Tyr701 and is essential for normal transcriptional activity of ISGF3 (interferon-stimulated gene factor 3) complex. It participates in the regulation of cytokine-signaling and cellular responses and plays an important role in growth and differentiation in a variety of cell types. This kit is designed for use with human cells and tissues.
      Catalogue NumberCBA035
      Brand Family Calbiochem®
      Application Data
      The sensitivity of this ELISA was compared to immunoblotting using known quantities of STAT1 (pTyr701). The data presented in Figure 1 show that the sensitivity of the ELISA is ~4x greater than that of immunoblotting. The bands shown in the immunoblotting data were developed using an anti STAT1 (pTyr701) 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). Distilled or deionized water. Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.). Data analysis and graphing software. 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
      ReferencesBoudny, V. and J. Kovarik 2002. Neoplasm 49, 349.0 Brivanlou, A.H. and J.E. Darnell 2002. Science 295, 813. Bromberg, J. 2002. J. Clin. Invest. 109, 1139. Levy, D.E. and J.E. Darnell 2002. Nat. Rev. Mol. Cell. Biol. 3, 651. Ghislain, J.J., et al. 2001. Interferon Cytokine Res. 21, 379. Shankaran, V. and R.D. Shreiber 2001. In Cytokine Reference. Volume 2: Receptors. J.J. Oppenheim, M. Feldmann, S.K. Durum, T. Hirano, J. Vilcek, and N.A. Nicola, editors. Academic Press, London, UK. pp.1819. Stephens, J.M., et al. 1998. J. Biol. Chem. 273, 31408.
      Product Information
      Form96 Tests
      Format96-well plate
      Kit containsSTAT1 (Tyr⁷⁰¹) Standard, Diluents, Detection Antibody, Secondary Antibody, Sample Buffer, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Assay range1.6-100 units/ml
      Assay time4 h
      Sample TypeCells
      Physicochemical Information
      Sensitivity< 0.9 Unit/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® PhosphoDetect™ STAT1 (pTyr⁷⁰¹) ELISA Kit is intended for the detection of STAT1 (pTyr⁷⁰¹) from lysates of human cells or tissues. This kit does not cross react with STAT1 from mouse or rat cells. For normalizing the STAT1 content of the samples, use the Calbiochem® STAT1 ELISA Kit (Cat. No. CBA034).
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsSTAT1 (Tyr⁷⁰¹) Standard, Diluents, Detection Antibody, Secondary Antibody, Sample Buffer, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Specifications

      Documentation

      References

      Reference overview
      Boudny, V. and J. Kovarik 2002. Neoplasm 49, 349.0 Brivanlou, A.H. and J.E. Darnell 2002. Science 295, 813. Bromberg, J. 2002. J. Clin. Invest. 109, 1139. Levy, D.E. and J.E. Darnell 2002. Nat. Rev. Mol. Cell. Biol. 3, 651. Ghislain, J.J., et al. 2001. Interferon Cytokine Res. 21, 379. Shankaran, V. and R.D. Shreiber 2001. In Cytokine Reference. Volume 2: Receptors. J.J. Oppenheim, M. Feldmann, S.K. Durum, T. Hirano, J. Vilcek, and N.A. Nicola, editors. Academic Press, London, UK. pp.1819. Stephens, J.M., et al. 1998. J. Biol. Chem. 273, 31408.
      User Protocol

      Revision20-October-2008 RFH
      Form96 Tests
      Format96-well plate
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C.
      Intended useThe Calbiochem® PhosphoDetect™ STAT1 (pTyr⁷⁰¹) ELISA Kit is intended for the detection of STAT1 (pTyr⁷⁰¹) from lysates of human cells or tissues. This kit does not cross react with STAT1 from mouse or rat cells. For normalizing the STAT1 content of the samples, use the Calbiochem® STAT1 ELISA Kit (Cat. No. CBA034).
      BackgroundSTAT1 is a member of the STAT family of proteins, which is comprised of STATs 1, 2, 3, 4, 5A, 5B, and 6. STAT homologs have also been described in Drosophila sp. and Dictyostellium discoidium. STAT family members are characterized by the presence of a DNA binding domain, an SH3 domain that mediates interaction with polyproline containing proteins, an SH2 domain that mediates interaction with phosphotyrosine containing proteins, and a C-terminal transactivation (TAD) domain. STAT proteins control the transcription of specific genes in response to cytokine stimulation. Alternative splicing yields two STAT1 isoforms, designated STAT1α and STAT1β, with MW=91 kDa and 84 kDa, respectively, which differ at their C-termini. As with all members of the STAT family, STAT1 exists in a latent state in the cytoplasm, and is activated and translocated to the nucleus in response to stimulation. Stimuli that activate STAT1 include interferons-α, and-γ, IL 6 family members including oncostatin M and LIF, as well as other cytokines, and the growth factor PDGF. The series of events leading to STAT1 activation arising from interferon-γ stimulation is best characterized. The interferon-γ receptor is composed of two IFNGR1 subunits and two IFNGR2 subunits that are localized at the plasma membrane as monomers prior to stimulation. Each IFNGR1 subunit has a constitutively associated JAK1, while each IFNGR2 has a constitutively associated JAK2. Upon interferon-γ stimulation, the dimeric ligand binds and dimerizes IFNGR1 subunits, which in turn recruit two IFNGR2 subunits through their extracellular domains. This association allows JAK1 and JAK2 to interact. The interacting JAK proteins transactivate one another by reciprocal tyrosine phosphorylation, and phosphorylate Tyr440 of the two IFNGR1 subunits contained in the receptor complex. These phosphorylated tyrosine residues on the IFNGR1 subunits provide paired docking sites for STAT1 via its SH2 domain. STAT1, recruited to the receptor complex, is then phosphorylated at tyrosine residue 701 by the JAKs. This tyrosine phosphorylation promotes STAT1's homo and heterodimerization mediated by reciprocal phosphotyrosine SH2 domain interaction and the dissociation of the dimers from the receptor complex. Activated STAT1 is then phosphorylated at Ser727 by an activity with MAPK like properties, and is translocated to the cell nucleus. STAT1 nuclear transport is GTP-dependent and involves STAT1's interaction with the protein importin. Once in the nucleus, STAT1-containing dimers complex with other nuclear proteins including p48, CBP, and p300. The STAT1-containing complexes regulate gene expression, either through interaction with the consensus DNA sequence TTCC(C or G)GAA (the GAS element [interferon-γ activated sequence]) located in the promoter regions of target genes, or through interacting with the interferon stimulated response element (ISRE). Genes that are upregulated in response to interferon-γ through STAT1 signaling are denoted immediate early genes, and include those for the transcription factor IRF-1, the type I Fcγ receptor, and guanylate binding protein 1. STAT1 activation of gene transcription is usually transient, being completed in about 15 min. A second wave of transcription, observed 6-8 h following STAT1 activation, produces intermediate gene transcripts by a process that requires additional protein synthesis. Intermediate genes include those for MHC class I and II. STAT1 is currently under investigation in many diverse areas of research including host responses to viral and bacterial infection, immune cell differentiation, and cell growth. STAT1 mediates apoptotic signals arising from interferon-γ stimulation, and may modulate anti apoptotic signaling arising from NFκB. STAT1 is also of interest in cancer studies, as this protein is constitutively activated in many tumors, including those of the breast, ovary, lung, head and neck, and in acute myeloid leukemia and erythroleukemia.
      Principles of the assayThe Calbiochem® PhosphoDetect™ STAT1 (pTyr⁷⁰¹) ELISA Kit is a solid phase sandwich Enzyme Linked Immuno Sorbent Assay (ELISA). A monoclonal antibody specific for STAT1 (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing STAT1 (pTyr⁷⁰¹), control specimens, and unknowns, are pipetted into these wells. During the first incubation, the STAT1 antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for STAT1 phosphorylated at Tyr⁷⁰¹ is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized STAT1 (pTyr⁷⁰¹) protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase labeled anti rabbit IgG (anti rabbit IgG HRP) is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the excess anti rabbit IgG HRP, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of STAT1 (pTyr⁷⁰¹) present in the original specimen.
      Materials provided• STAT1 (pTyr⁷⁰¹) Standard (Kit Component No. JA8074-1EA): 2 vials. Refer to vial label for quantity and reconstitution volume • Standard Diluent Buffer (Kit Component No. JA8075-25ML): 1 bottle 25 ml, Contains 15 mM sodium azide • STAT1-Antibody Coated Wells (Kit Component No. JA8076-1EA): 1 plate, 96 wells per plate • Rabbit anti-STAT1 (pTyr⁷⁰¹) (Detection Antibody) (Kit Component No. JA8077-11ML): 1 bottle, 11 ml, Contains 15 mM sodium azide • Goat Anti-rabbit IgG-Horseradish Peroxidase (HRP) Concentrate (Kit Component No. JA8078-125UL): 1 vial (100x), 0.125 ml, Contains 3.3 mM thymol • Sample Treatment Buffer II (Kit Component No. JA8079-10ML): 1 bottle, 10 ml • HRP Diluent (Kit Component No. JA8080-25ML): 1 bottle, 25 ml, Contains 3.3 mM thymol • Wash Buffer Concentrate (25x) (Kit Component No. JA8081-100ML): 1 bottle, 100 ml, • Soluble Substrate (Kit Component No. JA8082-25ML): 1 bottle, 25 ml, Tetramethylbenzidine (TMB) • Stop Solution (Kit Component No. JA8083-25ML): 1 bottle, 25 ml • Plate Sealers (Kit Component No. JA8084-EA): 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). Distilled or deionized water. Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.). Data analysis and graphing software. 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 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. Avoid multiple freeze-thaw cycles of frozen samples. 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 that are greater than the highest standard point should be diluted with Standard Diluent Buffer and retested. 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. Do not use reagents after the kit expiration date. 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. 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 of 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, 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 up to 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 Lysis Buffer is 24 h at 4°C. PMSF is very unstable and must be added prior to use, even if added previously. • Preparation of Cell Lysates: This protocol has been successfully applied to several cell lines. Researchers should optimize the cell lysate 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 cell pellet and expression of STAT1 (pTyr701). For example, 108 HeLa cells grown in DMEM plus 10% FBS and treated with 10 ng/ml IFN-γ for 20 min can be extracted in 1 ml of Lysis Buffer. Under these conditions, use of 1-10 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 STAT1 (pTyr701). 5. Transfer extract 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 Pre Treatment: Incubate each sample and control with an equal volume of Sample Treatment Buffer II on ice for 20 min. Dilute this mixture at least 5 fold in Standard Diluent Buffer. For example, for duplicate analyses, add 25 µl sample and 25 µl Sample Treatment Buffer II, then after incubation, add 200 µl Standard Diluent Buffer. The dilution chosen should be optimized for each experimental system.
      Reagent preparation• Reconstitution and Dilution of Human STAT1 (pTyr701) Standard: Note: This STAT1 (pTyr701) standard is prepared using purified, full-length, recombinant, phosphorylated STAT1 protein. One Unit of standard is equivalent to the amount of STAT1 (pTyr701) derived from 200 pg of STAT1 that was phosphorylated by activated JAK. 1. Reconstitute STAT1 (pTyr701) 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 STAT1 (pTyr701). Use the 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 STAT1 (pTyr701). 3. Make serial dilutions of the standard as described in the following dilution table. Mix thoroughly between steps. • Dilution of Human STAT1 (pTyr701) Standard

      Table 1: Dilution of Human STAT1 (pTyr701) Standard

      Remaining reconstituted standard should be discarded.

      Remaining reconstituted standard should be discarded. • 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 (100x concentrate) to reach room temperature. Gently mix. Pipette Anti rabbit IgG-HRP (100x 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. For Example:

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

      2. Return the unused Anti-rabbit IgG-HRP (100x 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. 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 of the Standard Diluent Buffer to zero wells. Well(s) reserved for chromogen blank should be left empty. 3. Add 100 µl of standards and pre treated diluted samples and controls (see page 14) to the appropriate microtiter wells. Tap gently on side of plate to thoroughly mix. (See Reagent Preparation, Section B.) 4. Cover wells with plate sealer and incubate for 2 h at room temperature 5. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing. 6. Pipette 100 ml of anti STAT1 (pTyr701) (Detection Antibody) solution into each well except the chromogen blank(s). Tap gently on the side of the plate to mix. 7. Cover wells 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 wells 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 Soluble 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 Abs values should be monitored and the substrate reaction stopped before the Abs of the positive wells exceed the limits of the instrument. The Abs 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 O.D., 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 STAT1 (pTyr701) concentrations for unknown samples and controls from the standard curve plotted in step 16. Multiply value(s) obtained for sample(s) by 2 to correct for the 1:2 dilution with Sample Treatment Buffer II and by the appropriate dilution factor to correct for the dilution with Standard Diluent Buffer. (Samples producing signals higher than the highest standard (100 Units/ml) should be further diluted in Standard Diluent Buffer and reanalyzed, multiplying the concentration by the appropriate dilution factor.)
      Standard curve

      Table 3: Typical Data Obtained with Standard

      The above data was obtained for the various standards over the range of 0 to 100 Units/ml STAT1 (pTyr701).

      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 difficult to obtain. Dilute samples >100 Units/ml with Standard Diluent Buffer; reanalyze 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 STAT1 or dephosphorylation of STAT1 (pTyr⁷⁰¹) in various matrices has not been investigated. Although STAT1 degradation or dephosphorylation of STAT1 (pTyr⁷⁰¹) in the Cell Lysis Buffer described in this protocol has not been seen to date, the possibility of this occurrence cannot be excluded.
      Sensitivity< 0.9 Unit/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <0.9 Units/ml of STAT1 (pTyr⁷⁰¹). This was determined by adding two standard deviations to the mean Abs obtained when the zero standard was assayed 30 times.

      Figure 1: Sensitivity

      The sensitivity of this ELISA was compared to immunoblotting using known quantities of STAT1 (pTyr701). The data presented in Figure 1 show that the sensitivity of the ELISA is ~4x greater than that of immunoblotting. The bands shown in the immunoblotting data were developed using an anti STAT1 (pTyr701) antibody and chemiluminescent detection.

      Assay Range1.6-100 units/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known STAT1 (pTyr701) 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, STAT1 (pTyr⁷⁰¹) Standard was spiked at 3 different concentrations into 10% Cell Lysis Buffer. The average recovery was 109%.
      Parallelism

      Figure 2: Parallelism

      Natural STAT1 (pTyr701) from IFN-γ-treated HeLa cell lysate was serially diluted in Standard Diluent Buffer. The optical density of each dilution was plotted against the STAT1 (pTyr701) standard curve. Parallelism demonstrated by the figure below indicated that the standard accurately reflects STAT1 (pTyr701) content in samples.

      Linearity

      Table 6: Linearity of Dilution

      HeLa cells were grown in tissue culture medium containing 10% fetal bovine serum, treated with 10 ng/ml IFN-γ for 20 min and lysed with Cell Lysis Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for STAT1 (pTyr701). Linear regression analysis of sample values versus the expected concentration yielded a correlation coefficient of 0.99.

      SpecificityThis STAT1 (pTyr⁷⁰¹) ELISA recognizes STAT1 of human origin and does not cross react with mouse or rat STAT1. This assay recognizes both alpha and beta isoforms of STAT1.

      Figure 3: Specificity

      HeLa cells were treated with IFN-γ at varying concentrations (0.001 to 20 ng/ml) for 20 min, then lysed. The level of phosphorylation at Tyr701 increases with dosage of IFN-γ. The results correlated very well with immunoblot analysis of the same samples (see insert in the graph below). Total STAT1 levels remain constant, while phospho levels increase with IFN-γ dose.

      Figure 4: Specificity

      The specificity of this assay for phosphorylated STAT1 (pTyr701) was confirmed by peptide competition. The data presented in Figure 4 show that the phosphopeptide containing the phosphorylated Tyr701 blocks the ELISA 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.