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CBA069 PhosphoDetect™ GSK-3β (pSer⁹) ELISA Kit

PhosphoDetect™ GSK-3β (pSer⁹) ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
CBA069
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      OverviewDetects and quantifies the level of GSK-3β (glycogen synthase kinase-3beta) phosphorylated at Ser9 in human, mouse, and rat cells. GSK-3β is a non-receptor serine/threonine kinase that is a key modulator of cell fate, neuronal plasticity, and tumorigenesis. It also plays an important role in diabetes, stroke, and Alzheimer's disease via regulation of microtubule stability and phosphorylation of the microtubule associating protein, tau.

      This product has been discontinued.
      Catalogue NumberCBA069
      Brand Family Calbiochem®
      Application Data
      The sensitivity of this ELISA was compared to immunoblotting using known quantities of GSK-3β (pSer9). The data show that the sensitivity of the ELISA is approximately 2x greater than that of immunoblotting. The bands shown in the immunoblot data were developed an anti-GSK-3β (pSer9) and chemiluminescent detection.

      Jurkat cells were grown in tissue culture medium containing 10% fetal calf serum were treated with 100 mM LiCl for 3 h and lysed with Cell Lysis Buffer. The lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for GSK-3β (pSer9). Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.
      Materials Required but Not Delivered• PBS
      • 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 Sample Preparation section)
      • Deionized or distilled H2O
      • Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.)
      • Data analysis and graphing software
      • Glass or plastic tubes for diluting and aliquoting standard
      • Absorbent paper towels
      • Calibrated beakers and graduated cylinders in various sizes
      References
      ReferencesBhatt, R.V., et al. 2004. J. Neurochem. 89, 1313.
      Kirshenboim, N., et al. 2004. J. Mol. Neurosci. 24, 237.
      Linseman, D.A., et al. 2004. J. Neurosci. 24, 9993.
      Cho, J.-H., et al. 2003. J. Biol. Chem. 278, 187.
      Doble, B.W. and Woodgett, J. R. 2003. J. Cell Sci. 116, 1175.
      Zhang, F., et al. 2003. J. Biol. Chem. 278, 33067.
      Martinez, A., et al. 2002. J. Med. Chem. 45, 1292.
      Ali, A., et al. 2001. Chem. Rev. 101, 2527.
      Godemann, R., et al. 1999. FEBS Lett. 454, 157.
      Product Information
      Unit of DefinitionOne unit (U) is defined as the amount of GSK-3β (pSer⁹) phosphorylated in 300 pg total GSK-3β.
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsGSK-3β (pSer⁹) Standard, Standard Diluent Buffer, GSK-3β Antibody-Coated 96-Well Plate, Rabbit Anti-GSK-3β (pSer⁹) Detector Antibody, Goat Anti-Rabbit IgG-HRP Concentrate, HRP Diluent, Wash Buffer Concentrate, TMB, Stop Solution, Plate Covers, and a user protocol.
      Applications
      Biological Information
      Assay range1.6-100 U/ml
      Assay time4 h
      Physicochemical Information
      Sensitivity< 0.4 U/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® GSK-3β (pSer⁹) ELISA Kit is designed to detect and quantify the level of GSK-3β protein phosphorylated at Ser⁹. This assay is intended for the detection of GSK-3β (pSer⁹) from lysates of human, mouse and rat cells. Calbiochem® also offers a GSK-3β ELISA Kit (Cat. No. CBA068), which quantifies GSK-3β regardless of phosphorylation status and allows normalization of phosphorylated GSK-3β to total GSK-3β.
      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 containsGSK-3β (pSer⁹) Standard, Standard Diluent Buffer, GSK-3β Antibody-Coated 96-Well Plate, Rabbit Anti-GSK-3β (pSer⁹) Detector Antibody, Goat Anti-Rabbit IgG-HRP Concentrate, HRP Diluent, Wash Buffer Concentrate, TMB, Stop Solution, Plate Covers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA069 0

      Documentation

      PhosphoDetect™ GSK-3β (pSer⁹) ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA069

      References

      Reference overview
      Bhatt, R.V., et al. 2004. J. Neurochem. 89, 1313.
      Kirshenboim, N., et al. 2004. J. Mol. Neurosci. 24, 237.
      Linseman, D.A., et al. 2004. J. Neurosci. 24, 9993.
      Cho, J.-H., et al. 2003. J. Biol. Chem. 278, 187.
      Doble, B.W. and Woodgett, J. R. 2003. J. Cell Sci. 116, 1175.
      Zhang, F., et al. 2003. J. Biol. Chem. 278, 33067.
      Martinez, A., et al. 2002. J. Med. Chem. 45, 1292.
      Ali, A., et al. 2001. Chem. Rev. 101, 2527.
      Godemann, R., et al. 1999. FEBS Lett. 454, 157.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      User Protocol

      Revision25-May-2010 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, mouse, rat
      StorageUpon arrival store the entire contents of the kit at 4°C.
      Intended useThe Calbiochem® GSK-3β (pSer⁹) ELISA Kit is designed to detect and quantify the level of GSK-3β protein phosphorylated at Ser⁹. This assay is intended for the detection of GSK-3β (pSer⁹) from lysates of human, mouse and rat cells. Calbiochem® also offers a GSK-3β ELISA Kit (Cat. No. CBA068), which quantifies GSK-3β regardless of phosphorylation status and allows normalization of phosphorylated GSK-3β to total GSK-3β.
      BackgroundGlycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, is one of two mammalian GSK-3 isoforms, designated GSK-3α (MW = 51 kDa) and GSK-3β (MW = 47 kDa). The two isoforms map to different genes: in humans, GSK-3α maps to 19q13.2, while GSK-3β maps to 3q13.3. The kinase domains of the two isoforms share the greatest homology (~95% homology), while the C-terminal regions are found to diverge between the two isoforms (36% homology). The two isoforms also differ slightly in structure. GSK-3α contains a glycine-rich extension in its N-terminus that is absent from GSK-3β, a feature that accounts for the difference in the molecular masses of the two isoforms. GSK-3β homologs appear to be ubiquitously expressed in eukaryotes. The Drosophila homolog is designated Shaggy/Zeste-white3. The GSKs were initially identified as negative regulators of glycogen synthase, a key enzyme in glycogen metabolism. Recent studies have revealed that GSK-3β also phosphorylates numerous other substrates, including β-catenin, CREB, p53, c-Myc, c-Jun, heat shock factor-1, cyclin D1, Bax, axin, the adenomatous polyposis coli (APC) gene product, eukaryotic protein synthesis initiation factor 2B (eIF-2B), and the microtubule associated proteins MAP1B and tau, suggesting important roles for this kinase in diverse cell functions. GSK-3β is phosphorylated at several sites. Phosphorylation of Tyr216, located in GSK-3β's activation loop, activates the enzyme, but may not be strictly required. Stimuli that enhance phosphorylation at Tyr216 include: staurosporine treatment, neurotrophic factor withdrawal with neuronal cell lines, lysophosphatidic acid (LPA) acting through the G proteins Gα12 and Gα13, and Ca2+ flux. Recent evidence implicate both Fyn and PYK2 in the phosphorylation of Tyr216. Phosphorylation of Ser9, located in the GSK-3β's N-terminal pseudosubstrate domain, inhibits enzyme activity. Phosphorylation of Ser9 produces a primed pseudosubstrate that binds to a positively charged pocket formed by arginine 96, arginine 180, and lysine 205, and competes with protein substrates. Stimuli that enhance phosphorylation of Ser9 include insulin, EGF, PDGF (through MAPKAP-K1), amino acids (through p70S6K), PKA activators, and PKC activators. Analysis of substrate specificity indicates that GSK-3's consensus sequence is Ser/Thr-X-X-X-Ser/Thr-P, where the N-terminal serine or threonine residue is the target for phosphorylation, X is any amino acid residue, but is often found to be proline, and the C-terminal serine or threonine is a residue that has undergone a priming phosphorylation by a different enzyme activity, such as PKA or CKI. This priming phosphorylation enhances the efficiency of phosphorylation of the target residues by GSK-3β by as much as 1000 fold. Crystallographic studies have revealed that the phosphorylated residue of primed substrates binds to GSK-3β's positively charged pocket, orienting the substrate within GSK-3β's catalytic groove. Consistent with this model, negatively charged residues can substitute for the phosphorylated serine or threonine of the primed substrate in enhancing the catalytic efficiency of GSK-3β. GSK-3β has been implicated in several signaling pathways, including the insulin, Wnt, and Hedgehog signaling pathways. In response to insulin, PI3K and AKT/PKB are activated, and in turn inhibit GSK-3β by enhancing phosphorylation at Ser9. The result is decreased phosphorylation of GSK-3β substrates including glycogen synthase and eIF-2B, and increased glycogen synthesis and protein synthesis. In Wnt signaling, GSK-3β is inhibited in a pathway that involves the protein, Dishevelled. Under resting conditions, active GSK-3β phosphorylates CKI-primed β-catenin, an event that targets β-catenin for ubiquitinylation and degradation by the proteasome. With the inactivation of GSK-3β in response to Wnt, β-catenin accumulates and translocates to the nucleus, where it influences gene expression. Hedgehog signaling similarly includes alterations in proteolysis of a GSK-3β consensus sequence-containing signaling protein designated Gli (the mammalian homolog of Cubitus interruptus [Ci] in Drosophila). As with β-catenin in the Wnt pathway, Gli also appears to play a role in transmitting signals into the cell nucleus. GSK-3β is currently under investigation in several important areas of research. Its interaction with the Wnt and Hedgehog pathways suggests a role in cell fate and morphology, and possibly the etiology of certain cancers. Its role in transducing the insulin signal has made it a target for study in type II diabetes. GSK-3β is also under investigation in several neurological disorders. GSK-3β is highly expressed in neurons and is found to phosphorylate the microtubule binding protein tau at sites that are abnormally phosphorylated in Alzheimer's disease. GSK-3β is also implicated in schizophrenia and bipolar disorder. For comparison of the level of phosphorylated and unphosphorylated GSK-3β in parallel, the GSK-3β ELISA Kit is also available (Cat. No. CBA068)
      Principles of the assayThe Calbiochem® PhosphoDetect™ GSK-3β (pSer⁹) Kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for GSK-3β (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing GSK-3β (pSer⁹), control specimens, and unknowns, are pipetted into these wells. During the first incubation, the GSK-3β (pSer⁹) antigen binds simultaneously to the immobilized (capture) antibody and to the solution phase rabbit polyclonal (detection) antibody specific for GSK-3β when phosphorylated at Ser⁹. After washing, 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 the second 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 GSK-3β (pSer⁹) present in the original specimen.
      Materials provided• GSK-3β (pSer⁹) Standard (Kit Component No. JA9282-1EA): 2 vials, refer to vial label for quantity and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA9283-25ML): 1 bottle, 25 ml, contains 0.1% sodium azide
      • GSK-3β Antibody-Coated 96-Well Plate (Kit Component No. JA9284-1EA): 1 plate, 96 wells supplied as twelve 8-well strips
      • Goat Anti-Rabbit IgG-HRP Concentrate (Kit Component No. JA9286-125UL): 1 vial, 125 μl, contains 3.3 mM thymol, supplied as 100X
      • HRP Diluent (Kit Component No. JA9287-25ML): 1 bottle, 25 ml, contains 3.3 mM thymol and yellow dye*
      • Wash Buffer Concentrate (Kit Component No. JA9288-100ML): 1 bottle, 100 ml, supplied as 25X
      • TMB (Kit Component No. JA9289-25ML): 1 bottle, 25 ml, ready-to-use
      • Stop Solution (Kit Component No. JA9290-25ML): 1 bottle, 25 ml, ready-to-use
      • Plate Cover (Kit Component No. JA9291-1EA): 3 adhesive strips
      • Rabbit Anti-GSK-3β (pSer⁹) Detector Antibody (Kit Component No. JA9285-6ML): 1 bottle, 6 ml, contains 0.1% sodium azide and blue dye*
      *In order to help our customers avoid any mistakes in pipetting, we provide colored Standard Diluent Buffer, Detection Antibody, and HRP Diluent to help monitor the addition of solution to the reaction well. This does not in any way interfere with the test results.
      Materials Required but not provided• PBS
      • 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 Sample Preparation section)
      • Deionized or distilled H2O
      • Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.)
      • Data analysis and graphing software
      • Glass or plastic tubes for diluting and aliquoting standard
      • Absorbent paper towels
      • Calibrated beakers and graduated cylinders in various sizes
      Precautions and recommendations• 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 stored at 4°C. All reagents should be warmed to room temperature before use.
      • Plates should be allowed to come to room temperature before opening the foil 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 frozen 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.
      • 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.
      • Read absorbance 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 TMB Substrate is light sensitive, avoid prolonged exposure to light. Also avoid contact between TMB 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 infectious agents.
      • Guidelines for Washing: Incomplete washing will adversely affect the results. 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 Diluted Wash Buffer. Let soak for 15 to 30 s and aspirate the liquid. Repeat as directed in the Detailed Protocol. Following the final wash, the plate should be inverted and tapped dry on absorbent tissue. Alternatively, the Diluted Wash Buffer may be put into a squirt bottle. If a squirt bottle is used, flood the plate with Diluted Wash Buffer, completely filling all wells. Following the final wash, the plate should be 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.
      PreparationRecommended Formulation of 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. Protocol for 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 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 Cell Lysis Buffer depends on the number of cells and expression of GSK-3β (pSer9). For example, 5 x 107 Jurkat cells grown in RPMI-1640 plus 10% FBS and treated with 100 mM LiCl for 2 h can be extracted in 1 ml of Cell Lysis Buffer. Under these conditions, use of 1-10 µl of the clarified cell lysate diluted to a volume of 50 µl/well in Standard Diluent Buffer (See Detailed Protocol) is sufficient for the detection of GSK-3β (pSer9). 5. Transfer lysates to microcentrifuge tubes and centrifuge at 13,000 rpm for 10 min at 4°C. 6. Aliquot the clear lysate to clean microcentrifuge 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 GSK-3β (pSer9) Standard Note: This GSK-3β (pSer9) standard is prepared using purified, full length, recombinant, phosphorylated GSK-3β protein expressed in Sƒ21 cells. One unit of standard is equivalent to the amount of GSK-3β (pSer9) phosphorylated in 300 pg of total GSK-3β protein. 1. Reconstitute GSK-3β (pSer9) 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 GSK-3β (pSer9). Use the standard within 1 h of reconstitution. 2. Add 0.15 ml of Standard Diluent Buffer to each of 6 tubes labeled 50, 25, 12.5, 6.25, 3.12 and 1.6 Units/ml GSK-3β (pSer9). 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 GSK-3β (pSer9) Standard

      • Storage and Final Dilution of Anti-Rabbit IgG Horseradish Peroxidase (HRP) Please Note: The Goat Anti-Rabbit IgG-HRP Concentrate is supplied in 50% glycerol, so the solution is viscous. To ensure accurate dilution, allow the Goat Anti-Rabbit IgG-HRP Concentrate to reach room temperature. Gently mix. Pipette the Goat 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 HRP Diluent for each 8-well strip used in the assay. Label as Anti-Rabbit IgG-HRP Working Solution. Use the following guidelines to determine the volume for the number of strips required for the assay.



      Table 2: Guidelines for diluting Anti-Rabbit IgG horseradish Peroxidase (HRP)

      2. Return the unused Goat Anti-Rabbit IgG-HRP Concentrate to the refrigerator. • Diluted Wash Buffer 1. Allow the Wash Buffer Concentrate to reach room temperature and mix to ensure that any precipitated salts are re-dissolved. Dilute 1 volume of the Wash Buffer Concentrate with 24 volumes 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 Diluted Wash Buffer. 2. Store both the concentrate and the Diluted 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. Return the unused strips to the pouch and store in the refrigerator for future use.
      2. Add 50 µl Standard Diluent Buffer to the zero wells. Well(s) reserved for the chromogen blank should be left empty.
      3. Add 50 µl standards, samples or controls to the appropriate wells. Tap gently on side of plate to mix.
      4. Add 50 µl Rabbit Anti-GSK-3β (pSer9) Detector Antibody. Samples prepared in Cell Lysis Buffer must be diluted 1:5 or greater in Standard Diluent Buffer (for example, 10 µl sample into 40 µl buffer). While a 1:5 sample dilution has been found to be satisfactory, higher dilutions such as 1:10 or 1:20 may be optimal. The dilution chosen should be optimal for each experimental system. Tap gently on side of plate to thoroughly mix.
      5. Cover the plate with a Plate Cover and incubate for 3 h at room temperature.
      6. Thoroughly aspirate or decant the solution from the wells and discard. Wash the wells 4 times. See Guidelines for Washing.
      7. Add 100 µl Goat Anti-Rabbit IgG-HRP Working Solution to each well except the chromogen blank(s).
      8. Cover plate with a Plate Cover and incubate for 30 min at room temperature.
      9. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Guidelines for Washing.
      10. Add 100 µl TMB to each well. The liquid in the wells will begin to turn blue.
      11. Incubate for 30 min at room temperature, in the dark. Please Note: Do not cover the plate with aluminum foil or metalized mylar. The incubation time for color development to occur is often determined by the plate reader used. Many plate readers have the capacity to record a maximum absorbance (Abs) of 2.0. The absorbance values should be monitored and the substrate reaction stopped before the absorbance of the positive wells exceeds the limits of the instrument. The absorbance values at 450 nm can only be read after the Stop Solution has been added to each well. If using a reader that records only to 2.0 absorbance points, stopping the assay after 20 to 25 min is suggested.
      12. Add 100 µl Stop Solution to each well. Tap side of plate gently to mix. The solution in the wells should change from blue to yellow.
      13. Read the absorbance of each well at 450 nm having blanked the plate reader against a chromogen blank composed of 100 µl each TMB and Stop Solution. Read the plate within 2 h of adding the Stop Solution.
      14. Plot 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.
      15. Read the GSK-3β (pSer9) concentrations for unknown samples and controls from the standard curve plotted in step 14. Multiply value(s) obtained for sample(s) by dilution factor to correct for the dilution with Standard Diluent Buffer. (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.)
      Example data

      Table 3: Example Data

      Example data obtained for the various diluted standards over the range of 0 to 100 Units/ml GSK-3β (pSer9).
      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; re-analyze 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 GSK-3β in various matrices has not been investigated. Although GSK-3β degradation or dephosphorylation of GSK-3β (pSer⁹) in the Cell Lysis Buffer described in this protocol has not been seen to date, the possibility of this occurrence cannot be excluded.
      Sensitivity< 0.4 U/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <0.4 Units/ml of human GSK-3β (pSer⁹). This was determined by adding two standard deviations to the mean absorbance obtained when the zero standard was assayed 30 times. In Jurkat cells stimulated by lithium chloride, this level of sensitivity was equivalent to the detection of GSK-3β (pSer⁹) in 5000 cells.

      Figure 1: Sensitivity

      The sensitivity of this ELISA was compared to immunoblotting using known quantities of GSK-3β (pSer9). The data show that the sensitivity of the ELISA is approximately 2x greater than that of immunoblotting. The bands shown in the immunoblot data were developed an anti-GSK-3β (pSer9) and chemiluminescent detection.
      Assay Range1.6-100 U/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known GSK-3β (pSer9) concentrations 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, GSK-3β (pSer⁹) Standard was spiked at 3 different concentrations into 50 µg/ml Jurkat cell lysate. The average recovery was 98.9%. High Dose Hook Effect Samples spiked with GSK-3β (pSer⁹) Standard up to 200 Units/ml give responses higher than that obtained from the highest standard point.
      Parallelism

      Figure 2: GSK-3β (pSer9) ELISA Parallelism

      Lithium chloride-treated Jurkat cells were lysed and the lysate was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the GSK-3β (pSer9) standard curve. Parallelism is demonstrated by the figure and indicates that the standard accurately reflects GSK-3β (pSer9) content in samples.
      Linearity

      Table 6: Linerearity of Dilution

      Jurkat cells were grown in tissue culture medium containing 10% fetal calf serum were treated with 100 mM LiCl for 3 h and lysed with Cell Lysis Buffer. The lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for GSK-3β (pSer9). Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.
      SpecificityThe PhosphoDetect™ GSK-3β (pSer⁹) ELISA Kit recognizes human, rat, and mouse. Other species have not been tested.

      Figure 3: Peptide Blocking

      The specificity of this assay for phosphorylated GSK-3β (pSer9) was confirmed by peptide competition. The data show that the phosphopeptide containing the phosphorylated Ser9 blocks the ELISA signal. Non-phosphorylated peptide does not block the signal.


      The PhosphoDetect™ GSK-3β (pSer⁹) ELISA Kit is specific for GSK-3β (pSer⁹). It does not cross-react with GSK-3α (pSer²¹).

      Figure 4: GSK-3β (pSer9] and GSK-3β

      Jurkat cells were treated with 100 mM LiCl for 2 h and untreated Jurkat cells were used as control. Cell extracts were prepared in 0.1% SDS extraction buffer, and analyzed using the GSK-3β (pSer9) ELISA and GSK-3β ELISA. The results show that the phosphorylation of GSK-3β (pSer9) is upregulated in LiCl-treated Jurkat cells, whereas the level of total GSK-3β remains consistant in LiCl-treated samples and untreated control.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Biosciences, Inc.
      Triton® is a registered trademark of Dow Chemical Company
      PhosphoDetect™ and Interactive Pathways™ are trademarks of EMD Biosciences, Inc.