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CBA066 PRAS40 ELISA Kit

PRAS40 ELISA Kit: Malzeme Güvenlik Bilgi Formu (MSDS) veya SDS, Analiz Sertifikası (COA) ve Kalite Uygunluk Sertifikası (COQ), dosyalar, broşürler ve diğer dokümanlar.
Detection Methods: Colorimetric
CBA066
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      Detects and quantifies the level of PRAS40 (Proline-Rich AKT Substrate of 40 kDa) independent of its phosphorylation state. PRAS40 is a 14-3-3 binding protein that is a direct substrate of Akt. It is believed to influence protein interactions, nuclear transport, and enzyme activities.
      Catalogue NumberCBA066
      Brand Family Calbiochem®
      Application Data
      The sensitivity of this ELISA was compared to immunoblotting using known quantities of PRAS40. 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 using a rabbit anti-PRAS40 antibody and chemiluminescent detection.
      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
      ReferencesSaito, A., et al. 2004. J. Neurosci. 24, 1584.
      Kovacina, K.S., et al. 2003. J. Biol. Chem. 278, 10189.
      Noshita, N., et al. 2003. Stroke 34, 1513.
      Cantley, L.C. 2002. Science 296, 1655.
      Lizcano, J.M. and Alessi, D.R. 2002. Curr. Biol. 12, R236.
      Macias, M.J., et al. 2002. FEBS Lett. 513, 30.
      Tzivion, G. and Avruch, J. 2002. J. Biol. Chem. 277, 3061.
      Cho, H., et al. 2001. J. Biol. Chem. 276, 38349.
      Noshita, N., et al. 2001. J. Cereb. Blood Flow Metabol. 21, 1442.
      Eves, E.M., et al. 1998. Mol. Cell Biol. 18, 2143.
      Fujio, Y., et al. 1999. Mol. Cell Biol. 19, 5073.
      Thorson, J.A., et al. 1998. Mol. Cell Biol. 18, 5229.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsPRAS40 Standard, Standard Diluent Buffer, PRAS40 Antibody-Coated 96-Well Plate, Rabbit Anti-PRAS40 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 range0.313-20 ng/ml
      Assay time4 h
      Physicochemical Information
      Sensitivity<200 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® PRAS40 ELISA Kit is designed to detect and quantify the level of PRAS40 protein independent of its phosphorylation state. This assay is intended for the detection of PRAS40 from lysate of mouse cells or tissues. This kit also recognizes human and rat PRAS40. This kit can be used to normalize the phosphorylated PRAS40 content of the samples when using the PhosphoDetect™ PRAS40 (pTyr²⁴⁶) ELISA Kit (Cat. No. CBA067).
      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 containsPRAS40 Standard, Standard Diluent Buffer, PRAS40 Antibody-Coated 96-Well Plate, Rabbit Anti-PRAS40 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
      Catalogue Number GTIN
      CBA066 0

      Documentation

      PRAS40 ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA066

      References

      Reference overview
      Saito, A., et al. 2004. J. Neurosci. 24, 1584.
      Kovacina, K.S., et al. 2003. J. Biol. Chem. 278, 10189.
      Noshita, N., et al. 2003. Stroke 34, 1513.
      Cantley, L.C. 2002. Science 296, 1655.
      Lizcano, J.M. and Alessi, D.R. 2002. Curr. Biol. 12, R236.
      Macias, M.J., et al. 2002. FEBS Lett. 513, 30.
      Tzivion, G. and Avruch, J. 2002. J. Biol. Chem. 277, 3061.
      Cho, H., et al. 2001. J. Biol. Chem. 276, 38349.
      Noshita, N., et al. 2001. J. Cereb. Blood Flow Metabol. 21, 1442.
      Eves, E.M., et al. 1998. Mol. Cell Biol. 18, 2143.
      Fujio, Y., et al. 1999. Mol. Cell Biol. 19, 5073.
      Thorson, J.A., et al. 1998. Mol. Cell Biol. 18, 5229.
      User Protocol

      Revision17-February-2010 RFH
      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® PRAS40 ELISA Kit is designed to detect and quantify the level of PRAS40 protein independent of its phosphorylation state. This assay is intended for the detection of PRAS40 from lysate of mouse cells or tissues. This kit also recognizes human and rat PRAS40. This kit can be used to normalize the phosphorylated PRAS40 content of the samples when using the PhosphoDetect™ PRAS40 (pTyr²⁴⁶) ELISA Kit (Cat. No. CBA067).
      BackgroundProline-Rich Akt Substrate of 40 kDa (PRAS40; accession numbers NP_115751 and NM_032375 for the protein and mRNA sequences, respectively) is a cytosolic protein with MW of 40 kDa that was identified and described in 2003. PRAS40 appears to be expressed by all tissues, with the highest levels in liver and heart. While most proteins contain on the order of 5% proline residues, PRAS40 is unique in containing a higher percentage of proline residues, ~15%, localized in proline-rich regions. PRAS40's proline rich regions may serve as binding sites for SH3 domain- and WW domain-containing proteins, possibly influencing the localization or function of these proteins. Several lines of evidence indicate that PRAS40 is a direct substrate for Akt. PRAS40 contains a consensus Akt phosphorylation site (RXRXXS/T) located at Thr246, a sequence that is conserved in other Akt substrates, including GSK-3β, BAD, and the mammalian homolog of the C. elegans transcription factor DAF-16. Recombinant PRAS40, produced in E. coli, can be phosphorylated in vitro by purified Akt. Further evidence was obtained with an HA epitope tagged version of PRAS40, expressed transiently in HEK293 cells, that produced a phosphorylated band by Western blotting using an antibody directed to the Akt substrate consensus sequence. The phosphorylation of this band was enhanced when the cells were co-transfected with a constitutively activated Akt mutant. Finally, PRAS40 phosphorylation was decreased in cells lacking Akt1 and Akt2. Within the Akt phosphorylation site consensus sequence of PRAS40 is the consensus sequence for binding by 14-3-3 proteins (RXXpS/pT). Direct evidence for phosphorylated PRAS40's binding to 14-3-3 proteins was revealed with in vitro binding assays, and with immunoprecipitation experiments in which c-myc tagged PRAS40 constructs were precipitated with c myc antibody, then probed with a 14-3-3 antibody in immunoblotting. The 14-3-3 proteins comprise a family of evolutionarily conserved scaffolding and adaptor proteins that modulate numerous signaling events within cells by influencing protein:protein interactions, nuclear transport, and enzyme activities. These proteins play a role in regulating apoptosis by preventing the interaction of BAD and Bcl-2 following Akt activation. Studies with wortmannin and LY294002 lend support for the existence of a phosphoinositide 3 kinase (PI3K), Akt, PRAS40, and 14-3-3-containing signaling module. Wortmannin, an inhibitor of PI3K, reduced the phosphorylation state of PRAS40 in several experimental systems. This inhibition could be prevented by expression of a constitutively activated Akt mutant. Treatment of cells with LY294002, another PI3K inhibitor, prevented PRAS40's binding to 14-3-3 proteins. Several stimuli enhance the level of PRAS40 phosphorylation, including insulin, NGF, and PDGF. PRAS40's roles in conferring resistance to apoptosis, regulating cell growth, regulating glucose uptake, and in the etiology of diabetes are currently under investigation. PRAS40 is also of interest in stroke. In transient cerebral focal ischemia, a model for stroke, overexpression of phosphorylated PRAS40 was found to attenuate apoptosis.
      Principles of the assayThe Calbiochem® PRAS40 ELISA Kit is a solid phase sandwich Enzyme Linked Immuno Sorbent Assay (ELISA). A monoclonal antibody specific for PRAS40 (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing PRAS40, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the PRAS40 antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for PRAS40 is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized PRAS40 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 PRAS40 present in the original specimen.
      Materials provided• PRAS40 Standard (Kit Component No. JA9252): 2 vials, please refer to the vial label for reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA9253): 1 bottle, 25 ml, contains 15 mM sodium azide and red dye*
      • PRAS40 Antibody-Coated 96-Well Plate (Kit Component No. JA9254): 1 plate, 96 wells supplied as twelve 8-well strips
      • Anti-Rabbit IgG-HRP Concentrate (Kit Component No. JA9256): 1 vial, 125 µl, supplied as 100X, contains 3.3 mM thymol
      • HRP Diluent (Kit Component No. JA9257): 1 bottle, 25 ml, contains 3.3 mM thymol and yellow dye*
      • Wash Buffer Concentrate (Kit Component No. JA9258): 1 bottle, 100 ml, supplied as 25X
      • TMB (Kit Component No. JA9259): 1 bottle, 25 ml, ready-to-use
      • Stop Solution (Kit Component No. JA9260): 1 bottle, 25 ml, ready-to-use
      • Plate Cover (Kit Component No. JA9261): 3 adhesive strips
      • Rabbit Anti-PRAS40 Detector Antibody (Kit Component No. JA9255): 1 bottle, 11 ml, contains 15 mM sodium azide and blue dye*
      * In order to help 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.
      • Extracted cell lysate samples containing PRAS40 protein should be diluted with Standard Diluent Buffer at least 1:10. 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 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 is light sensitive, avoid prolonged exposure to light. Also avoid contact between TMB 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 Concentrate 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 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 number of cells and expression of PRAS40. For example, 4 x 106 3T3L1 cells grown in DMEM plus 10% FBS can be extracted in 1 ml Cell Lysis Buffer. Under these conditions, use of 1-10 µl 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 PRAS40. 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. Protocol for Preparation of Tissue Extracts This protocol has been successfully applied to several tissue types. Some optimization may be required for each specific application. 1. Add protease inhibitors to the Tissue Extraction Buffer just prior to use. 2. Weigh the tissue sample. 3. Add 10 ml Tissue Extraction Buffer per 1 g tissue. 4. Homogenize. 5. Centrifuge the sample at 10,000 rpm for 5 min to pellet the tissue debris. 6. Collect supernatant, aliquot and freeze at 80°C. Avoid multiple freeze thaw cycles.
      Reagent preparation• Reconstitution and Dilution of PRAS40 Standard Note: The PRAS40 standard is prepared from purified, full length, recombinant, PRAS40 protein expressed in Sf9 cells. 1. Reconstitute PRAS40 Standard with Standard Diluent Buffer. Refer to standard vial label for instructions and reconstitution volume. Swirl or mix gently and allow to sit for 10 min to ensure complete reconstitution. Label as 20 ng/ml PRAS40. Use the standard within 1 h of reconstitution. 2. Add 0.25 ml Standard Diluent Buffer to each of 6 tubes labeled 5, 2.5, 1.25, 0.62, and 0.313 ng/ml PRAS40. 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: Reconstitution and Dilution of PRAS40 Standard

      • Storage and Final Dilution of Anti-Rabbit IgG 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 Goat Anti-Rabbit IgG-HRP Concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper. 1. Add 10 µl Anti-Rabbit IgG-HRP Concentrate to 1 ml HRP Diluent. One ml is sufficient for an 8-well strip. 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 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 100 µl of standards, samples or controls to the appropriate wells. Standards, samples, and controls will have a red color. 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. The dilution chosen should be optimized for each experimental system. Tap gently on side of plate to thoroughly mix.
      4. Cover wells with a Plate Cover 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 Guidelines For Washing.
      6. Add 100 µl of Rabbit Anti-PRAS40 Detector Antibody to each well except the chromogen blank(s). This solution will have a blue color. Tap gently on the side of the plate to mix.
      7. Cover wells with a Plate Cover 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 Guidelines For Washing.
      9. Add 100 µl Anti-Rabbit IgG-HRP Working Solution to each well except the chromogen blank(s). This solution will have a yellow color.
      10. Cover wells with a Plate Cover 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 Guidelines For Washing.
      12. Add 100 µl of TMB 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 TMB 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 exceed the limits of the instrument. The absorbance values at 450 nm can only be read after the Stop Solution has been added to each well. If using a reader that records only to absorbance of 2.0, stopping the assay after 20 to 25 min is suggested.
      14. 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.
      15. 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 after adding the Stop Solution.
      16. 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.
      17. Read the PRAS40 concentrations for unknown samples and controls from the standard curve plotted in step 16. Multiply value(s) obtained for sample(s) by the dilution factor to correct for the dilution in step 3. (Samples producing signals higher than the highest standard (20 ng/ml) should be further diluted in Standard Diluent Buffer and reanalyzed, multiplying the concentration by the appropriate dilution factor.)
      Example data

      Table 3: Typical Results from the Standard

      Data obtained for the various standards over the range of 0 to 20 ng/ml PRAS40.
      Limitations of the assayDo not extrapolate the standard curve beyond the 20 ng/ml standard point; the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute samples >20 ng/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 PRAS40 in various matrices has not been investigated.
      Sensitivity<200 pg/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <200 pg/ml of PRAS40. This was determined by adding two standard deviations to the mean absorbance obtained when the zero standard was assayed 30 times. Using 3T3L1 cells, this level of sensitivity was equivalent to the detection of PRAS40 in 4000 cells.












      Figure 1: Sensitivity

      The sensitivity of this ELISA was compared to immunoblotting using known quantities of PRAS40. 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 using a rabbit anti-PRAS40 antibody and chemiluminescent detection.
      Assay Range0.313-20 ng/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known PRAS40 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, PRAS40 Standard was spiked at 3 different concentrations into 5% Cell Lysis Buffer. The percent recovery was calculated as an average of 84%.
      Parallelism

      Figure 2: Parallelism

      Natural PRAS40 from 3T3L1 cell lysate was serially diluted in Standard Diluent Buffer. The optical density of each dilution was plotted against the PRAS40 standard curve. Parallelism was demonstrated by the figure below and indicated that the standard accurately reflects PRAS40 content in samples.
      Linearity

      Table 6: Linerearity of Dilution

      3T3L1 cells were grown in DMEM containing 10% fetal bovine serum at 37°C and lysed with Cell Lysis Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for PRAS40 content. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.
      Specificity

      Figure 3: Detection of PRAS40 in Various Cell Lines and Mouse Tissues

      The PRAS40 ELISA Kit is specific for the measurement of total PRAS40 protein. To determine the specificity of this ELISA kit, cell lysates from different cell lines, each at a final concentration of 20 µg/ml total protein, were analyzed. The data show that the kit detects PRAS40 in cell lysates from mouse 3T3L1, rat L6, and human Jurkat. PRAS40 was also detected in tissue extracts from mouse heart, liver, and lung. The levels of PRAS40 protein detected with this ELISA kit are consistent with results obtained by immunblot analysis.
      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.