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CBA065 PhosphoDetect™ IκBα (pSer³²) ELISA Kit

PhosphoDetect™ IκBα (pSer³²) 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.
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      Descripción

      Replacement Information

      Tabla espec. clave

      Detection Methods
      Colorimetric
      Description
      Overview

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      This product has been discontinued.



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      Detects and quantifies the level of IκBα protein phosphorylated at Ser32 in human cells. IκBα belongs to IκB protein family and is known to modulate NF-kB (nuclear factor-κB) nuclear translocation. It plays an important role in immune and inflammatory responses, cell division and apoptosis.
      Catalogue NumberCBA065
      Brand Family Calbiochem®
      Application Data
      Known quantities of IκBα (pSer32) protein were measured as outlined in the Detailed Protocol and by immunoblotting using an anti-IκBα (pSer32) primary antibody and chemiluminescent detection. The ELISA is approximately 2x more sensitive than immunoblotting.

      The specificity of this assay for IκBα phosphorylated at Ser32 was confirmed by peptide competition. The data show that only the phosphopeptide containing the phosphorylated Ser32 could block the ELISA signal in Jurkat cells treated with TNF-α. The same sequence containing non-phosphorylated serine at position 32 did not block the signal. A peptide conatining a phosphoserine at position 36 also did not block the signal.

      Cell lysates made from Jurkat cells were harvested at various times following treatment with TNF-α and compared to non-treated controls, as determined using the Detailed Protocol provided. The data presented here also demonstrate that there is an initial increase in phosphorylation followed by a reduction in the level of Total IκBα which is attributable to the TNF-α-stimulated degradation of IκBα. The results correlate well with immunoblot analysis.


      Samples of known IκBα (pSer32) concentration were assayed in replicates of 16 to determine precision within an assay.

      Samples were assayed 48 times in multiple assays to determine precision between assays.
      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
      ReferencesMa, M.H., et al. 2003. Clin. Cancer Res. 9, 1136.
      Ghosh, S. and Karin, M. 2002. Cell 109, S81.
      Adams, J. 2001. Semin. Oncol. 28, 613.
      Berenson, J.R., et al. 2001. Semin. Oncol. 28, 626.
      Joyce, D., et al. 2001 Cytokine Growth Factor Rev. 12, 73.
      Tanaka K., et al. 2001. Biochimie 83, 351.
      Israel, A. 2000. Trends Cell Biol. 10, 129.
      Malek, S., et al. 1998. J. Biol. Chem. 273, 25427.
      Product Information
      Unit of DefinitionOne unit is defined as the amount of IκBα pSer³² derived from 40 pg IκBα phosphorylated by IKKα.
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsIκBα (pSer³²) Standard, Standard Diluent Buffer, IκBα Antibody-Coated 96-Well Plate, Rabbit Anti-IκBα (pSer³²) Detector Antibody, 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 Units/ml
      Assay time4 h
      Physicochemical Information
      Sensitivity< 1.0 Units/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® PhosphoDetect™ Human IκBα (pSer³²) ELISA Kit is designed to detect and quantify the levels of IκBα protein that is phosphorylated at Ser³². This ELISA kit can be used to detect IκBα in human cells. This kit is not recommended for detection of mouse and rat IκBα. This assay is intended for the detection of IκBα (pSer³²) from cell lysates.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsIκBα (pSer³²) Standard, Standard Diluent Buffer, IκBα Antibody-Coated 96-Well Plate, Rabbit Anti-IκBα (pSer³²) Detector Antibody, Anti-Rabbit IgG-HRP Concentrate, HRP Diluent, Wash Buffer Concentrate, TMB, Stop Solution, Plate Covers and a user protocol.
      Specifications
      Global Trade Item Number
      Número de referencia GTIN
      CBA065 0

      Documentation

      PhosphoDetect™ IκBα (pSer³²) ELISA Kit Certificados de análisis

      CargoNúmero de lote
      CBA065

      Referencias bibliográficas

      Visión general referencias
      Ma, M.H., et al. 2003. Clin. Cancer Res. 9, 1136.
      Ghosh, S. and Karin, M. 2002. Cell 109, S81.
      Adams, J. 2001. Semin. Oncol. 28, 613.
      Berenson, J.R., et al. 2001. Semin. Oncol. 28, 626.
      Joyce, D., et al. 2001 Cytokine Growth Factor Rev. 12, 73.
      Tanaka K., et al. 2001. Biochimie 83, 351.
      Israel, A. 2000. Trends Cell Biol. 10, 129.
      Malek, S., et al. 1998. J. Biol. Chem. 273, 25427.

      Folleto

      Cargo
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protocolo de usuario

      Revision18-September-2008 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, not mouse, not rat
      StorageUpon arrival store the entire contents of the kit at 4°C.
      Intended useThe Calbiochem® PhosphoDetect™ Human IκBα (pSer³²) ELISA Kit is designed to detect and quantify the levels of IκBα protein that is phosphorylated at Ser³². This ELISA kit can be used to detect IκBα in human cells. This kit is not recommended for detection of mouse and rat IκBα. This assay is intended for the detection of IκBα (pSer³²) from cell lysates.
      BackgroundIκBα (Inhibitor of Nuclear Factor (NF)-κB α isoform) is a member of the IκB protein family with a MW of 40 kDa. IκBα is ubiquitously expressed among mammals. Located at IκBα's N-terminus is a signal response domain which contains serine residues that can be phosphorylated, while the C-terminus contains a PEST domain, a common feature among proteins with high turnover rates. As with all members of the IκB family, IκBα possesses ankyrin repeats approximately 33 amino acids in length, which mediate binding to the Rel homology region of NF-κB. The interaction of IκBα with NF-κB masks the nuclear localization sequence of NF-κB, preventing NF-κB translocation to the nucleus. A variety of stimuli can activate gene expression by liberating NF-κB through the degradation of IκBα. These stimuli include the proinflammatory cytokines TNF-α and IL-1β, chemokines, PMA, growth factors, LPS, UV irradiation, and viral infection, as well as various chemical and physical stresses. The series of events leading to this liberation is well-defined. In response to stimulus, Ser32/36 of IκBα are phosphorylated, which provides a signal for IκBα E3 ligase, a protein complex composed of SKP-1, Cul-1, Roc1, and Fbw1. IκBα E3 ligase polyubiquitinates IκBα at lysine residues 21 and 22, and the polyubiquitinated IκBα is then targeted to the 26S proteasome for degradation. Liberated NF-κB is transported across the nuclear membrane, where it activates transcription by binding to the consensus sequence GGRNNYYCC*, which is found in the promoter regions of a large number of genes including IL-6, VEGF, VCAM-1, ICAM-1, HIV long terminal repeat, and many others. The initial event which targets IκBα for degradation is phosphorylation of Ser32/36. This phosphorylation is catalyzed by a protein complex known as IKK (IκB kinase). IKK contains two kinase subunits designated IKKα (MW=85 kDa) and IKKβ (MW=87 kDa), and scaffold protein designated IKKγ/NEMO (MW=48 kDa). The kinetic properties of IKK are regulated by complex formation as well as by phosphorylation events catalyzed by upstream kinases including members of the MAPK cascade, the SAPK/JNK cascade and NIK. Through its regulation of NF-κB, IκBα controls immune and inflammatory responses, cell division, and apoptosis. Numerous disease states including arthritis, asthma, and inflammatory bowel disease are associated with loss of IκBα regulation. The importance of regulation of IκBα in cancer is underscored by the observation that multiple myelomas often possess polymorphisms at IκBα regulatory sites, and certain symptoms can be ameliorated using the proteasome inhibitor PS-341 which favors the sequestration of NF-κB in the cytoplasm.
      Principles of the assayThe Calbiochem® PhosphoDetect™ Human IκBα (pSer³²) Kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for human IκBα (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing IκBα (pSer³²), control specimens, and unknowns, are pipetted into these wells. During the first incubation, the IκBα antigen binds to the immobilized (capture) antibody. After washing, an antibody specific for IκBα (pSer³²) is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized IκBα 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 IκBα (pSer³²) present in the original specimen.
      Materials provided• IκBα (pSer³²) Standard (Kit Component No. JA9242): 2 vials, refer to vial label for quantity and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA9243): 1 bottle, 25 ml, contains 15 mM sodium azide and red dye*
      • IκBα Antibody-Coated 96-Well Plate (Kit Component No. JA9244): 1 plate, 96 wells supplied as twelve 8-well strips
      • Anti-Rabbit IgG-HRP Concentrate (Kit Component No. JA9246): 1 vial, 125 μl, contains 3.3 mM thymol, supplied as 100X
      • HRP Diluent (Kit Component No. JA9247): 1 bottle, 25 ml, contains 3.3 mM thymol and yellow dye*
      • Wash Buffer Concentrate (Kit Component No. JA9248): 1 bottle, 100 ml supplied as 25X
      • TMB (Kit Component No. JA9249): 1 bottle, 25 ml, ready-to-use
      • Stop Solution (Kit Component No. JA9250): 1 bottle, 25 ml, ready-to-use
      • Plate Cover (Kit Component No. JA9251): 3 adhesive strips
      • Rabbit Anti-IκBα (pSer³²) Detector Antibody (Kit Component No. JA9245): 1 bottle, 6 ml, contains 15 mM sodium azide and blue dye*

      * In order to help avoid mistakes in pipetting during the prodcedure, we provide colored Standard Diluent Buffer, Detection Antibody, and HRP Diluent to help monitor the addition of reagents to the wells. These do not interfere with the assay in any way.
      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.
      • Extracted cell lysate samples containing human IκBα (pSer32) 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 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 of Cell Lysis Buffer depends on the number of cells and expression of IκBα and phosphorylation of IκBα. For example, 108 Jurkat cells treated with 20 ng/ml TNF-α for 5 min can be extracted in 1 ml of Cell Lysis Buffer. Under these conditions, use of 0.4-4 ml of the clarified cell lysate diluted to a volume of 100 µl/well in Standard Diluent Buffer (See Detailed Protocol) is sufficient for the detection of IκBα (pSer32). 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 IκBα Standard Note: The IκBα (pSer32) Standard was prepared using purified, full length, human recombinant IκBα expressed in E. coli. One Unit of standard is defined as the amount of IκBα (pSer32) derived from 40 pg of IκBα, which was phosphorylated by IKKα. Subsequent lots of standard will be normalized to this lot of material to allow consistency of IκBα (pSer32) quantitation. 1. Reconstitute the IκBα (pSer32) 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 IκBα. 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 IκBα (pSer32). 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 IκBα (pSer32) Standard

      • Storage and Final Dilution of Anti-Rabbit IgG Horseradish Peroxidase (HRP) Please Note: The Anti-Rabbit IgG-HRP Concentrate is supplied in 50% glycerol, so the solution is viscous. To ensure accurate dilution, allow the Anti-Rabbit IgG-HRP Concentrate to reach room temperature. Gently mix. Pipette Anti-Rabbit IgG-HRP Concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper. 1. Within 1 h of use, 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 Anti-Rabbit IgG-HRP Concentrate to the refrigerator. • Diluted Wash Buffer 1. Allow the Wash Buffer Concentrate 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 100 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 designated wells. Standards, samples, and controls will have a red color. 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.
      4. Add 50 µl Rabbit Anti-IκBα (pSer32) Detector Antibody to each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
      5. Cover plate with a Plate Cover and incubate for overnight (14-18 h) at 4°C.
      6. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Guidelines for Washing.
      7. Add 100 µl Anti-Rabbit IgG-HRP Working Solution to each well except the chromogen blank(s).
      8. Cover the plate with the 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. 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 IκBα (pSer32) 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.)
      Example data




      Table 3: Example Data

      Example data were obtained for the various diluted standards over the range of 0 to 100 Units/ml IκBα (pSer32).
      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 IκBα in various matrices has not been investigated.
      Sensitivity< 1.0 Units/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <1.0 Units/ml of human IκBα (pSer³²). This was determined by adding two standard deviations to the mean absorbance obtained when the zero standard was assayed 30 times. In Jurkat cells cultured in complete medium, this sensitivity corresponded to the IκBα protein extractable from 4000 Jurkat cells treated with 20 ng/ml TNF-α for 5 min.

      Figure 1: Sensitivity

      Known quantities of IκBα (pSer32) protein were measured as outlined in the Detailed Protocol and by immunoblotting using an anti-IκBα (pSer32) primary antibody and chemiluminescent detection. The ELISA is approximately 2x more sensitive than immunoblotting.
      Assay Range1.6-100 Units/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known IκBα (pSer32) 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, recombinant human IκBα (pSer³²) was spiked into the lysate at 3 different concentrations in 20% Cell Lysis Buffer and the percent recovery over endogenous levels calculated. On average, 98.2% recovery was observed.
      Parallelism












      Figure 2: IκBα (pSer32) ELISA Parallelism

      Natural phospho IκBα from human Jurkat cell lysates was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the IκBα (pSer32) standard curve. Parallelism was demonstrated by the figure above and indicated that the standard accurately reflects IκBα (pSer32) content in samples.
      Linearity

      Table 6: Linerearity of Dilution

      Jurkat cells were grown in tissue culture medium containing 10% fetal calf serum treated with TNF-α for 5 min and lysed with Cell Lysis Buffer. The lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for IκBα (pSer32) content. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99 in both cases.
      SpecificityThe PhosphoDetect™ IκBα (pSer³²) ELISA Kit is specific for measurement of human IκBα that is phosphorylated at Ser³². The reactivity of this ELISA with other IκB isoforms has not been established. This ELISA kit does not react with mouse and rat IκBα.




      Figure 3: Assay Specificity

      The data indicate that IκBα (pSer32) recognizes E. coli-derived recombinant IκBα phosphorylated in vitro by recombinant IKKα subunit. An ELISA measuring total IκBα recognizes both phosphorylated and non-phosphorylated IκBα.


      Figure 4: Peptide Competition Assay

      The specificity of this assay for IκBα phosphorylated at Ser32 was confirmed by peptide competition. The data show that only the phosphopeptide containing the phosphorylated Ser32 could block the ELISA signal in Jurkat cells treated with TNF-α. The same sequence containing non-phosphorylated serine at position 32 did not block the signal. A peptide conatining a phosphoserine at position 36 also did not block the signal.


      Figure 5: Detection of IκBα in Jurkat Cells

      Cell lysates made from Jurkat cells were harvested at various times following treatment with TNF-α and compared to non-treated controls, as determined using the Detailed Protocol provided. The data presented here also demonstrate that there is an initial increase in phosphorylation followed by a reduction in the level of Total IκBα which is attributable to the TNF-α-stimulated degradation of IκBα. The results correlate well with immunoblot analysis.
      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.