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	48-602MAG	Buffer Detection Kit for Magnetic Beads	1 Kit

CBA038 PhosphoDetect™ Insulin Receptor (pTyr^1162/1163) ELISA Kit

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

CoA
References
Brochures
User Protocol

Detection Methods: Colorimetric

Recommended Products

Overview

Replacement Information

Key Spec Table

Detection Methods
Colorimetric

Description
Overview	This product has been discontinued. Detects and quantifies the levels of insulin receptor (IR) that are phosphorylated at Tyr^1162/1163. The IR is a cell surface receptor that belongs to the superfamily of the growth factor receptor tyrosine kinases and regulates multiple signaling pathways. It is activated by two separate events, where three residues, Tyr¹¹⁵⁸, Tyr¹¹⁶² and Tyr¹¹⁶³, located in the regulatory loop, appear to play a major role. Although this kit is designed for use with human cell lines, platelets and lymphocytes, it cross-reacts with mouse and rat cells.
Catalogue Number	CBA038
Brand Family	Calbiochem®
Application Data	The sensitivity of this ELISA was compared to immunoblotting using cell lysate with known quantities IR (pTyr^1162/1163). The data presented in Figure 1 show that the sensitivity of the ELISA is about 2x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using rabbit anti- IR (pTyr^1162/1163), 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, p. 12). • Distilled or deionized H₂O. • 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
References	Bevan, P., 2001. J. Cell. Sci. 114, 1429. Skorey, K.I., et al. 2001. Anal. Biochem. 291, 269. Ottensmeyer, F.P., et al. 2000. Biochemistry 39, 12103. Playford, M.P., et al. 2000. Proc. Nat'l. Acad. Sci. USA 97, 12103. Virkamaki, A., et al. 1999. J. Clin. Invest. 103, 931. Wei, L., et al. 1995 . J. Biol. Chem. 270, 8122. Ebina, Y., et al. 1985. Cell 40, 747. Ullrich, A., et al. 1985. Nature 313, 756.

References

Bevan, P., 2001. J. Cell. Sci. 114, 1429.
Skorey, K.I., et al. 2001. Anal. Biochem. 291, 269.
Ottensmeyer, F.P., et al. 2000. Biochemistry 39, 12103.
Playford, M.P., et al. 2000. Proc. Nat'l. Acad. Sci. USA 97, 12103.
Virkamaki, A., et al. 1999. J. Clin. Invest. 103, 931.
Wei, L., et al. 1995 . J. Biol. Chem. 270, 8122.
Ebina, Y., et al. 1985. Cell 40, 747.
Ullrich, A., et al. 1985. Nature 313, 756.

Product Information
Unit of Definition	One unit is defined as the amount of IR (pTyr<sup>1162/1163</sup>) derived from 0.6 ng of IR (β-subunit) in CHO-T cells stimulated with 100 nM insulin.
Detection method	Colorimetric
Form	96 Tests
Format	96-well plate
Kit contains	IR (pTyr^1162/1163) Standard, Diluents, Detector Antibody, Secondary Antibody, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
Positive control	Human Insulin Receptor (β-subunit)

Applications

Biological Information
Assay range	1.6-100 Units/ml
Assay time	4 h
Sample Type	Cells

Physicochemical Information
Sensitivity	<0.8 Units/ml

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS

Safety Information

Product Usage Statements
Intended use	The Calbiochem^® Phospho-Insulin Receptor (pTyr^1162/1163) ELISA is designed to detect and quantify the levels of insulin receptor that are phosphorylated at tyrosine residues 1162 and 1163 of insulin receptor. Both natural (heterotetrameric) and recombinant IR react in this assay. This ELISA is not cross-reactive with IGF-1R and allows a differentiation of the phosphorylation and activation of IR from that of IGF-1R. Although this ELISA kit is developed using human cells, cross-reactivity with mouse and rat insulin receptor is documented. This assay is intended for detection of IR (pTyr^1162/1163) from lysates of cells. For normalizing the IR content of the samples, an IR (β-subunit) ELISA kit which is independent of phosphorylation status is available from Calbiochem^® (Cat. No. CBA039)

Product Usage Statements

Intended use

The Calbiochem^® Phospho-Insulin Receptor (pTyr^1162/1163) ELISA is designed to detect and quantify the levels of insulin receptor that are phosphorylated at tyrosine residues 1162 and 1163 of insulin receptor. Both natural (heterotetrameric) and recombinant IR react in this assay. This ELISA is not cross-reactive with IGF-1R and allows a differentiation of the phosphorylation and activation of IR from that of IGF-1R. Although this ELISA kit is developed using human cells, cross-reactivity with mouse and rat insulin receptor is documented. This assay is intended for detection of IR (pTyr^1162/1163) from lysates of cells. For normalizing the IR content of the samples, an IR (β-subunit) ELISA kit which is independent of phosphorylation status is available from Calbiochem^® (Cat. No. CBA039)

Storage and Shipping Information
Ship Code	Blue Ice Only
Toxicity	Multiple Toxicity Values, refer to MSDS
Storage	+2°C to +8°C
Storage Conditions	Upon arrival store the entire contents of the kit at 4°C.
Do not freeze	Ok to freeze

Packaging Information

Transport Information

Supplemental Information
Kit contains	IR (pTyr^1162/1163) Standard, Diluents, Detector Antibody, Secondary Antibody, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.

Specifications

Global Trade Item Number
Catalogue Number	GTIN
CBA038	0

Documentation

PhosphoDetect™ Insulin Receptor (pTyr^1162/1163) ELISA Kit Certificates of Analysis

Title	Lot Number
CBA038	Enter Lot Number

References

Reference overview
Bevan, P., 2001. J. Cell. Sci. 114, 1429. Skorey, K.I., et al. 2001. Anal. Biochem. 291, 269. Ottensmeyer, F.P., et al. 2000. Biochemistry 39, 12103. Playford, M.P., et al. 2000. Proc. Nat'l. Acad. Sci. USA 97, 12103. Virkamaki, A., et al. 1999. J. Clin. Invest. 103, 931. Wei, L., et al. 1995 . J. Biol. Chem. 270, 8122. Ebina, Y., et al. 1985. Cell 40, 747. Ullrich, A., et al. 1985. Nature 313, 756.

Reference overview

Brochure

Title
Protein Kinase Assay and Detection Kits Brochure

User Protocol

Revision	17-June-2010 RFH
Form	96 Tests
Format	96-well plate
Detection method	Colorimetric
Species	human, mouse, rat
Storage	Upon arrival store the entire contents of the kit at 4°C.
Intended use	The Calbiochem^® Phospho-Insulin Receptor (pTyr^1162/1163) ELISA is designed to detect and quantify the levels of insulin receptor that are phosphorylated at tyrosine residues 1162 and 1163 of insulin receptor. Both natural (heterotetrameric) and recombinant IR react in this assay. This ELISA is not cross-reactive with IGF-1R and allows a differentiation of the phosphorylation and activation of IR from that of IGF-1R. Although this ELISA kit is developed using human cells, cross-reactivity with mouse and rat insulin receptor is documented. This assay is intended for detection of IR (pTyr^1162/1163) from lysates of cells. For normalizing the IR content of the samples, an IR (β-subunit) ELISA kit which is independent of phosphorylation status is available from Calbiochem^® (Cat. No. CBA039)
Background	Insulin receptor (IR), a cell surface receptor, binds insulin and mediates its action on target cells. Insulin receptor belongs to the superfamily of the growth factor receptor tyrosine kinases that regulate multiple signaling pathways through activation of a series of phosphorylation cascades. The insulin receptor is a heterotetrameric membrane glycoprotein consisting of disulfide-linked subunits in a β-α-α-β configuration. The α-subunit (135 kDa) is completely extracellular, whereas the β-subunit (95 kDa) possesses a single transmembrane domain with tyrosine kinase activity. Insulin binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Once activated, the IR initiates a variety of metabolic functions including glucose transport, glycogen synthesis, protein synthesis, translational control and mitogenesis. Defects in IR signaling pathway result in insulin resistance and thus high blood glucose associated with type II/non-insulin-dependent diabetes. The IR also plays an important role in neurological function and in hypertension. Important down-stream proteins associated with IR signaling include IRS-1, JAK1, JAK2, STAT1, STAT3, PI3 kinase, GSK-3β, PKC, SHC, ERK1/2 and many others. Insulin receptor and insulin-like growth factor 1 receptor (IGF-1R), which are structurally related, share conserved tyrosine residues that are phosphorylated in response to insulin and in IGF-1, respectiviely. Functionally, the insulin receptor regulates metabolism and IGF-1R mediates growth and differentiation. The catalytic loops within the tyrosine kinase domains of the IR/IGF-1R share the same residue sequence with a three-tyrosine motif corresponding to Tyr^{1158/1162/1163} (for IR) and Tyr^{1131/1135/1136} (for IGF-1R). It is generally believed that autophosphorylation within the activation loop proceeds in a progressive manner initiating at the second tyrosine (1162 or 1135), followed by phosphorylation at the first tyrosine (1158 or 1131), then the last (1163 or 1136), upon which the IR or IGF-1R becomes fully active.
Principles of the assay	The Calbiochem^® PhosphoDetect™ Insulin Receptor (pTyr^1162/1163) ELISA Kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for IR (β-subunit) (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing IR (pTyr^1162/1163), control specimens and unknowns, are pipetted into these wells. During the first incubation, the IR antigen binds to the immobilized (capture) antibody. After washing, an antibody specific for IR phosphorylated at Tyr^1162/1163 is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized IR 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 IR (pTyr^1162/1163) present in the original specimen.
Materials provided	• Human IR (pTyr^1162/1163) Standard (Kit Component No. JA8095-1EA): (2 vials) Refer to vial label for quantity and reconstitution volume • Standard Diluent Buffer (Kit Component No. JA8096-25ML): 1 bottle, 25 ml, Contains ≤0.1% sodium azide • IR (β-subunit) Antibody-Coated Wells (Kit Component No. JA8097-1EA): 1 plate, 96 wells per plate • Rabbit anti-(pTyr^1162/1163) (Detection Antibody) (Kit Component No. JA8098-11ML): 1 bottle, 11 ml, Contains ≤0.1% sodium azide • Rabbit Anti-IR IgG-Horseradish Peroxidase (HRP) Concentrate (Kit Component No. JA8099-125UL): 1 vial (100x), 0.125 ml, Contains 3.3 mM thymol • HRP Diluent (Kit Component No. JA8100-25ML): 1 bottle, 25 ml, Contains 3.3 mM thymol • Wash Buffer Concentrate (25x) (Kit Component No. JA8101-100ML): 1 bottle, 100 ml • Soluble Substrate (Kit Component No. JA8102-25ML): 1 bottle, 25 ml, Tetramethylbenzidine (TMB) • Stop Solution (Kit Component No. JA8103-25ML): 1 bottle, 25 ml • Plate Sealers (Kit Component No. JA8104-1EA): 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, p. 12). • Distilled or deionized H₂O. • 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 containing IR (pTyr^1162/1163) 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. • 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. • 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 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. • Preparation of Cell Lysates: This protocol has been applied to several cell lines using the Cell Lysis Buffer above. Researchers should optimize the cell lysis protocol 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 IR (pTyr^1162/1163). 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.
Reagent preparation	• Reconstitution and Dilution of IR (pTyr^1162/1163) Standard: Note: This IR (pTyr^1162/1163) standard is a lyophilized lysate from insulin-stimulated human IR transfected CHO cells (CHO-T). 1 Unit of standard is equivalent to the amount of IR (pTyr^1162/1163) derived from 0.6 ng of IR (β-subunit) in CHO-T cells stimulated with 100 nM insulin. Subsequent lots of standard will be normalized to this lot of material to allow consistency of IR (pTyr^1162/1163) quantitation. 1. Reconstitute IR (pTyr^1162/1163) 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 IR (pTyr^1162/1163). 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 IR (pTyr^1162/1163). 3. Make serial dilutions of the standard as described in the following dilution table. Mix thoroughly between steps. • Dilution of IR (pTyr^1162/1163) Standard: Table 1: Dilution of IR (pTyr^1162/1163) Standard Discard all remaining reconstituted and diluted standards after completing assay. Return the Standard Diluent Buffer to the refrigerator. • 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 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 protocol	Be 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 to the appropriate wells. Samples prepared in Cell Lysis Buffer must be diluted 1:10 or greater in Standard Diluent Buffer (for example, 10 µl sample into 90 µl buffer). While a 1:10 sample dilution has been found to be satisfactory, higher dilutions such as 1:25 or 1:50 may be optimal for sample resolution. The dilution chosen should be optimized for each investigation's system. 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. 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-IR (pTyr^1162/1163) (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 an 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 Abs, 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 IR (pTyr^1162/1163) 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 (100 Units/ml) should be further diluted in Standard Diluent Buffer and re-analyzed, multiplying the concentration found by the appropriate dilution factor.) 18. Values of IR (pTyr^1162/1163) may be normalized for IR (β-subunit) content by parallel measurement with the Calbiochem^® Insulin Receptor (β-subunit) ELISA Kit (Cat. No. CBA039).
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 IR (pTyr^1162/1163).
Limitations of the assay	Do 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 lysis buffers has not been thoroughly investigated. The rate of degradation of native IR (pTyr^1162/1163) in various matrices has not been investigated. Although IR (pTyr^1162/1163) degradation in the Cell Lysis Buffer described in this protocol has not been seen to date, the possibility of this occurrence cannot be excluded.
Sensitivity	<0.8 Units/ml
Sensitivity Notes	The analytical sensitivity of this assay is <0.8 Units/ml of IR (pTyr^1162/1163). 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 cell lysate with known quantities IR (pTyr^1162/1163). The data presented in Figure 1 show that the sensitivity of the ELISA is about 2x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using rabbit anti- IR (pTyr^1162/1163), and chemiluminescent detection.
Assay Range	1.6-100 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.
Recovery	The recovery of IR (pTyr^1162/1163) added to 100 µg/ml of a Jurkat cell lysate in Cell Lysis Buffer (followed by a 1:10 dilution in Standard Diluent Buffer) averaged 104%.
Parallelism	Figure 2: Parallelism Natural IR (pTyr^1162/1163) from lysate of insulin-stimulated CHO-T cells was serially diluted in Standard Diluent Buffer. The optical density of each dilution was plotted against the human IR (pTyr^1162/1163) standard curve. Parallelism was demonstrated by the figure below and indicated that the Standard accurately reflects IR (pTyr^1162/1163) content in samples.
Linearity	Table 6: Linearity of Dilution Lysate Buffer was spiked with IR (pTyr^1162/1163) and serially diluted in Standard Diluent Buffer over the range of the assay. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.
Specificity	Figure 3: Specificity This IR (pTyr^1162/1163) ELISA kit is specific for measurement of IR that is dually phosphorylated at Tyr^1162/1163. IR of natural or recombinant (β-subunit) origin is reactive in this assay. This kit detects phosphorylated IR in insulin-stimulated CHO-T cells and does not detect non-phosphorylated IR in unstimulated cells, as shown in figure 3. Figure 4: Specificity The specificity of this assay for IR phosphorylated at Tyr^1162/1163 was confirmed by peptide competition. Phosphorylated IR was quantitated in the assay as usual except that the detection antibody was preincubated with IR-derived peptides at a concentration of 0.1-1 µg/ml. The data presented in Figure 3 show that only the peptide corresponding to the region surrounding Tyr^1162/1163, containing the phospho-tyrosines, could block the ELISA signal. The peptides containing phosphorylated tyrosine at positions 972 and 1158 did not block the signal. Figure 5: Specificity Figure 5 shows that IR (pTyr^1162/1163) phosphorylation in CHO-T cells is dependent on levels of insulin stimulation. Cells (~90% confluent) were treated with insulin at varying concentrations (0-100 nM) for 10 min, lysed and quantitated in parallel for IR content (both β-subunit and pTyr^1162/1163). The amount of IR (β-subunit) remains constant, while the level of phosphorylation at Tyr^1162/1163 decreases with diminishing insulin dosage. Phosphorylation at IR pTyr¹¹⁵⁸, shows a similar pattern.
Registered Trademarks	Calbiochem^® is a registered trademark of EMD Chemicals, Inc. Triton^® is a registered trademark of Dow Chemical Company PhosphoDetect™ and InteractivePathways™ are trademarks of EMD Chemicals, Inc.