Millipore Sigma Vibrant Logo

CBA005 PhosphoDetect™ Akt (pSer⁴⁷³) ELISA Kit

PhosphoDetect™ Akt (pSer⁴⁷³) 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.

Sinónimos: Protein Kinase B, Phospho-Specific (Ser⁴⁷³) ELISA Kit

Detection Methods: Colorimetric
CBA005
  
Recuperando precio...
No pudo obtenerse el precio
La cantidad mínima tiene que ser múltiplo de
Maximum Quantity is
Al finalizar el pedido Más información
Ahorró ()
 
—
Solicitar precio
—
Disponibilidad a confirmar Disponibilidad a confirmar
Ingrese cantidad 
Suspendido
Cantidades limitadas disponibles
Debe confirmarse disponibilidad
    El resto: se avisará
      El resto: se avisará
      Se avisará
      Póngase en contacto con el Servicio de Atención al Cliente
      Contact Customer Service

       

      Póngase en contacto con el Servicio de Atención al Cliente

      Descripción

      Replacement Information

      Tabla espec. clave

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      This product has been discontinued.



      We apologize for the inconvenience, but we do not currently have an alternative product.






      Detects Akt1 when phosphorylated on Ser473. Does not detect phosphorylated Akt3. Cross-reactivity with phosphorylated Akt2 has not been thoroughly evaluated. Akt is activated by a variety of stimuli including insulin through phosphorylation on Thr308 and Ser473. Once phosphorylated Akt migrates to the nucleus where it is involved in a variety of cellular processes such as the transduction of signals that prevent apoptosis. Although this kit was developed for human samples, it has been found to cross react with mouse and rat.
      Catalogue NumberCBA005
      Brand Family Calbiochem®
      SynonymsProtein Kinase B, Phospho-Specific (Ser⁴⁷³) ELISA Kit
      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 multi-channel pipette is desirable for large assays
      • Cell Lysis Buffer (see recommended formulation below)
      • Deionized or distilled H2O
      • Automated or manual plate washer such as a squirt bottle or a manifold dispenser
      • Linear, log-log, or semi-log graph paper
      • Glass or plastic tubes for diluting and aliquoting standard
      • Absorbent paper towels
      • Calibrated beakers and graduated cylinders in various sizes
      References
      ReferencesKops, G.J., et al. 2002. Mol. Cell Biol. 22, 2025.
      Stiles, B., et al. 2002. Mol. Cell Biol. 22, 3842.
      Sun, M., et al. 2001. Am. J. Pathol. 159, 431.
      Fulton, D., et al. 1999. Nature 399, 597.
      Datta, S.R., et al. 1997. Cell 91, 231.
      Alessi, D.R., et al. 1996. EMBO J. 15, 6541.
      Bellacosa, A., et al. 1995. Int. J. Cancer 64, 280.
      Burgering, B.M. and Coffer, P.J. 1995. Nature 376, 599.
      Franke, T.F., et al. 1995. Cell 81, 727.
      Bellacosa, A., et al. 1993. Oncogene 8, 745.
      Staal, S.P. 1987. Proc. Natl. Acad. Sci. USA 84, 5034.
      Coffer, P.J. and Woodgett, J.R. 1991. Eur. J. Biochem. 201, 475.
      Jones, P.F., et al. 1991. Proc. Natl. Acad. Sci. USA 88, 4171.
      Product Information
      Unit of DefinitionOne unit is defined as the amount of Akt (pSer⁴⁷³) derived from 100 pg of Akt, which was phosphorylated by MAPKAP2 and PDK1.
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, Akt Phospho-Ser⁴⁷³ Standard, Diluents, Detector Antibody, Secondary Antibody, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Positive controlAkt (pSer⁴⁷³)
      Applications
      Biological Information
      Assay range1.6-100 units/ml
      Assay time4 h
      Sample TypeCell lysates
      Physicochemical Information
      Sensitivity≤0.8 units/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 21/22-36/38

      Harmful in contact with skin and if swallowed.
      Irritating to eyes and skin.
      S PhraseS: 26-36/37-45

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing and gloves.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Intended useThe Akt ELISA kit is designed to detect and quantify the level of Akt protein that is phosphorylated at serine residue 473. Although performance characterization of this ELISA kit was done primarily on human cell lines, cross-reactivity of this kit with mouse and rat cells was observed. This assay is intended for the detection of Akt (pSer⁴⁷³) 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.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, Akt Phospho-Ser⁴⁷³ Standard, Diluents, Detector Antibody, Secondary Antibody, Wash Buffer, TMB Substrate, Stop Solution, Plate Covers, and a user protocol.
      Specifications
      Global Trade Item Number
      Número de referencia GTIN
      CBA005 0

      Documentation

      PhosphoDetect™ Akt (pSer⁴⁷³) ELISA Kit Certificados de análisis

      CargoNúmero de lote
      CBA005

      Referencias bibliográficas

      Visión general referencias
      Kops, G.J., et al. 2002. Mol. Cell Biol. 22, 2025.
      Stiles, B., et al. 2002. Mol. Cell Biol. 22, 3842.
      Sun, M., et al. 2001. Am. J. Pathol. 159, 431.
      Fulton, D., et al. 1999. Nature 399, 597.
      Datta, S.R., et al. 1997. Cell 91, 231.
      Alessi, D.R., et al. 1996. EMBO J. 15, 6541.
      Bellacosa, A., et al. 1995. Int. J. Cancer 64, 280.
      Burgering, B.M. and Coffer, P.J. 1995. Nature 376, 599.
      Franke, T.F., et al. 1995. Cell 81, 727.
      Bellacosa, A., et al. 1993. Oncogene 8, 745.
      Staal, S.P. 1987. Proc. Natl. Acad. Sci. USA 84, 5034.
      Coffer, P.J. and Woodgett, J.R. 1991. Eur. J. Biochem. 201, 475.
      Jones, P.F., et al. 1991. Proc. Natl. Acad. Sci. USA 88, 4171.

      Folleto

      Cargo
      Akt
      Biologics 31.1
      Diabetes and Obesity
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protein Kinase Assay and Detection Kits Brochure
      Protocolo de usuario

      Revision02-July-2008 RFH
      SynonymsProtein Kinase B, Phospho-Specific (Ser⁴⁷³) ELISA Kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, mouse, rat
      StorageUpon arrival store the entire contents of the kit at 4°C.
      Intended useThe Akt ELISA kit is designed to detect and quantify the level of Akt protein that is phosphorylated at serine residue 473. Although performance characterization of this ELISA kit was done primarily on human cell lines, cross-reactivity of this kit with mouse and rat cells was observed. This assay is intended for the detection of Akt (pSer⁴⁷³) from cell lysates.
      BackgroundAkt, also known as the protein kinase B-α (PKB-α) or RAC-PKα, was initially identified as one of the downstream targets of PI-3 Kinase (PI3-K). Akt is now known to consist of three highly conserved isoforms, which are designated in humans as Akt1, Akt2, and Akt3. Each isoform consists of an N-terminus pleckstrin homology (PH) domain, which mediates lipid-protein or protein-protein interactions, and a C-terminus kinase catalytic domain. Although each kinase is expressed differentially in a tissue-specific manner, they respond in a similar fashion to various stimuli. Akt can be activated by a diverse array of growth factors and physiologic stimuli in a PI3-K-dependent manner. Activation of Akt kinase is a multi-step process involving both membrane translocation and phosphorylation. Activated PI3-K generates 3' phosphoinositide products, 3,4,5-triphosphates (PI-3,4,5-P3) and PI-3,4-P2. Akt is recruited from the cytosol to the plasma membrane through the interaction of its PH domain with these phosphoinositides. Upon membrane localization, Akt undergoes a conformational change, which makes it accessible to phosphorylation at Thr308 and Ser473 in the kinase domain by PDK-1 and related kinases. Activated Akt then acts as a key mediator of signals for cell survival, proliferation, angiogenesis, and a number of metabolic effects of insulin. The effects of Akt activation may be mediated by modulation of expression or activity of various molecules including Bcl-2, BAD, caspase-9, endothelial nitric oxide synthase (eNOS), glycogen synthase, and transcription factors (NF-kB, Forkhead, CREB, Mdm2). Because of its growth-promoting effects, Akt is also emerging as a central player in tumorigenesis. A number of oncogenes and tumor suppressor genes act upstream of Akt to influence cancer progression. Deletion of PTEN, a tumor suppressor gene that encodes a phosphatase, correlates with increased Akt activity in several cancers. Similarly, overexpression of active Ras, Her/Neu, or Akt genes causes hyperactivation of Akt in many cancers including pancreatic, gastric, breast, ovarian and prostate adenocarcinomas.
      Principles of the assayThe Akt (pSer⁴⁷³) kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for Akt1 (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing Akt (pSer⁴⁷³), control specimens, and unknowns, are pipetted into these wells. During the first incubation, the Akt antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody, specific for Akt phosphorylated at Ser⁴⁷³, is added to the wells. During the second incubation, this antibody serves as a detector by binding to the immobilized Akt 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-part sandwich. After a third incubation and washing to remove 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 Akt (pSer⁴⁷³) present in the original specimen.
      Materials provided• Akt (pSer⁴⁷³) Standard (Kit Component No. JA7780): 2 vials of lyophilized Akt standard, please refer to vial label for formulation and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA7781):: 25 ml of buffer containing 15 mM sodium azide
      • AKT Antibody Coated Plate (Kit Component No. JA7782):: One 96-well plate coated with Akt antibody
      • Detector Antibody (Kit Component No. JA7783):: 11 ml of rabbit anti-Akt (pSer⁴⁷³) antibody containing 15 mM sodium azide
      • Secondary Antibody (Kit Component No. JA7784):: 125 µl of anti-rabbit IgG-Horseradish Peroxidase (HRP) Concentrate, (100X) supplied in 50% glycerol containing 3.3 mM thymol
      • Secondary Antibody Diluent (Kit Component No. JA7785):: 25 ml of diluent containing 3.3 mM thymol
      • Wash Buffer Concentrate (Kit Component No. JA7786):: 100 ml of 25X wash buffer
      • TMB Substrate (Kit Component No. JA7787):: 25 ml of Tetramethylbenzidine
      • Stop Solution (Kit Component No. JA7788):: 25 ml
      • Plate Covers (Kit Component No. JA7789):: 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 multi-channel pipette is desirable for large assays
      • Cell Lysis Buffer (see recommended formulation below)
      • Deionized or distilled H2O
      • Automated or manual plate washer such as a squirt bottle or a manifold dispenser
      • Linear, log-log, or semi-log graph paper
      • Glass or plastic tubes for diluting and aliquoting standard
      • Absorbent paper towels
      • Calibrated beakers and graduated cylinders in various sizes
      Precautions and recommendations• When not in use, kit components should be refrigerated. All reagents should be warmed to room temperature before use.
      • AKT antibody coated 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 Akt (pSer473) protein extracted from cells 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. The SDS concentration resulting from the cell extraction buffer should be less than 0.01% before adding to the plate.
      • When pipetting reagents, maintain a consistent order of addition from well-to-well to ensure 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 stabilized chromogen is light sensitive, avoid prolonged exposure to light. Avoid contact between stabilized chromogen and metal or color may develop.
      • Note: This kit contains materials with small quantities of sodium azide, which 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.
      • Washing Procedure: Incomplete washing will adversely affect the test outcome. All washing must be performed with the wash buffer supplied with the kit. To wash the plate completely aspirate the liquid from all wells by gently lowering an aspiration tip or 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 assay procedure. After washing the plate should be inverted and tapped dry on absorbent paper towels. Note: If a squirt bottle is used, flood the plate with wash buffer to completely fill all wells. After washing the plate should be inverted and gently tapped dry on absorbent paper towels. If using an automated washer follow the operating instructions for the washing equipment. 30 s soak cycles should be programmed into the wash cycle.
      PreparationCell Lysate Note: While this protocol has been successfully applied to several cell lines of human, mouse or rat origin, researchers should optimize the cell extraction 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 extraction buffer depends on the cell number in cell pellet and expression of Akt (pSer473). For example, 4 x 107 Jurkat cells grown in RPMI plus 10% FBS can be extracted in 1 ml of lysate buffer. Under these conditions, use of 0.1-5 µl of the clarified cell extract diluted to a volume of 100 µl/well in standard diluent buffer (see assay procedure) is sufficient for the detection of Akt (pSer473). 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. Reconstitution and Dilution of Akt (pSer473) Standard Note: This Akt (pSer473) standard is prepared using purified, full length, human recombinant Akt expressed in Sf21 cells. One unit of standard is defined as the amount of Akt (pSer473) derived from 100 pg of Akt, which was phosphorylated by MAPKAP2 and PDK1. 1. Reconstitute Akt (pSer473) standard with standard diluent buffer according to standard vial label. Swirl or mix gently and allow to sit for 10 min to ensure complete reconstitution. Label as 100 units/ml Akt (pSer473). 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 Akt (pSer473). 3. Make serial dilutions of the standard as described in the following dilution table. Mix thoroughly between steps.

      Table 1: Dilution of Akt (pSer473) Standard

      The remaining reconstituted standard should be discarded or frozen at -80°C. The standard can be frozen and thawed one time without loss of immunoreactivity.

      Anti-Rabbit IgG Horseradish Peroxidase • Note: The anti-rabbit IgG-HRP 100X concentrate is supplied in 50% glycerol. To ensure accurate dilution, allow the viscous anti-rabbit IgG-HRP concentrate to reach room temperature. Mix gently and pipette anti-rabbit IgG-HRP concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper. • Dilute 10 µl of this 100X concentrated solution with 1 ml of HRP diluent for each 8-well strip used. This is the anti-rabbit IgG-HRP working solution, and it should be used within 1 h. 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 (for example, 50 ml may be diluted up to 1.25 liters, 100 ml may be diluted up to 2.5 liters). This is the working wash buffer. • Store the concentrate and the working wash buffer in the refrigerator. The diluted buffer should be used within 14 days.
      Reagent preparationCell 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 Na3O4V 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, reconstituted and added according to manufacturer's instructions (~250 µl/5 ml Cell Lysis Buffer) Note: This buffer is stable for up to 3 weeks at 4°C or for up to 6 months when aliquoted (without protease inhibitors and PMSF added) and stored at -20°C. When stored frozen the Cell Lysis Buffer should be thawed on ice. 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 just prior to use, even if added previously.
      Detailed protocolAllow 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, samples, or controls to the appropriate wells. Samples prepared in Cell Lysis Buffer must be diluted at least 1:10 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
      4. May be optimal. The dilution chosen should be optimized for each experimental system. Tap gently on the side of plate to thoroughly mix. (See reagent preparation.)
      5. Cover the wells with a plate cover and incubate for 2 h at room temperature or overnight at 4°C.
      6. Thoroughly aspirate or decant the solution from the wells and discard the liquid. Wash wells 4 times as described previously.
      7. Pipette 100 µl of anti-Akt (pSer473) detector antibody solution into each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
      8. Cover the wells with a plate cover and incubate for 1 h at room temperature.
      9. Thoroughly aspirate or decant the solution from the wells and discard the liquid. Wash wells 4 times.
      10. Add 100 µl of anti-rabbit IgG-HRP working solution to each well except the chromogen blank(s).
      11. Cover the wells with a plate cover and incubate for 30 min at room temperature.
      12. Thoroughly aspirate or decant the solution from the wells and discard the liquid. Wash wells 4 times.
      13. Add 100 µl of stabilized chromogen to each well. The liquid in the wells will begin to turn blue.
      14. Incubate for 30 min at room temperature and in the dark. Note: Do not cover the plate with aluminum foil or
      15. metalized mylar. The incubation time for chromogen substrate is often determined by the plate reader used. Many plate readers have the capacity to record a maximum absorbance (Abs) of 2.0. The Abs values should be monitored and the substrate reaction stopped before the Abs of the positive wells exceed the limits of the instrument. The Abs values at 450 nm can be read only 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.
      16. Add 100 µl of Stop Solution to each well. Tap the side of plate gently to mix. The solution in the wells should change from blue to yellow.
      17. Read the absorbance of each well at 450 nm having blanked the plate reader against a chromogen blank composed of 100 µl each of stabilized chromogen and stop solution. Read the plate within 2 h after adding the stop solution.
      18. Plot on graph paper the absorbance of the standards against the standard concentration. The background absorbance should 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.
      19. Read the Akt (pSer473) concentrations for unknown samples and controls from the standard curve plotted in step 16.
      20. 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 (100 units/ml) should be further diluted in standard diluent Buffer and re-analyzed, multiplying the concentration by the appropriate dilution factor.
      Assay characteristics and examples

      Table 2: Typical Data

      The above data was obtained for the various standards over the range of 0 to 100 units/ml of Akt (pSer473).
      Limitations of the assayDo not extrapolate the standard curve beyond 100 units/ml, as the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute these samples with standard diluent buffer and multiply the results by the appropriate dilution factor.

      While the influence of various extraction buffers has not been thoroughly investigated, Akt degradation or dephosphorylation of Akt (pSer⁴⁷³) in the cell extraction buffer described in this protocol has not yet been seen.
      Sensitivity≤0.8 units/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <0.8 units/ml of Akt (pSer⁴⁷³). This was determined by adding two standard deviations to the mean Abs obtained when the zero standard was assayed 30 times. The sensitivity of this ELISA was compared to immunoblotting using known quantities of Akt (pSer⁴⁷³). In Jurkat cells cultured under optimal conditions, this sensitivity corresponded to the Akt (pSer⁴⁷³) extract from 4000 cells.

      Figure 1: Sensitivity

      The data presented in figure 1 shows that the sensitivity of the ELISA is ~2X greater than that of immunoblotting. The bands shown in the immunoblot were developed using Anti-Akt, Phospho-Specific (Ser473), (Cat. No. 124003), an alkaline phosphatase conjugated anti-rabbit IgG analyzed by chemiluminescent substrate, and autoradiography.

      Assay Range1.6-100 units/ml
      Precision

      Table 3: Intra-Assay Precision

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


      Table 4: Inter-Assay Precision

      Samples were assayed 48 times in multiple assays to determine precision between assays.
      RecoveryTo evaluate recovery, unstimulated mouse cell extracts were prepared in Cell Extraction Buffer and adjusted to 200 µg/ml total protein. Recombinant Akt (pSer⁴⁷³) at 3 levels were spiked into the cell extract and percent recovery calculated over endogenous levels. On average, 93% recovery was observed.
      Parallelism

      Figure 2: Parallelism

      Natural Akt (pSer473) from extracts of Jurkat cells cultured in RPMI + 10% FCS were serially diluted in standard diluent buffer. The optical density of each dilution was plotted against the Akt (pSer473) standard curve. Parallelism demonstrated in figure 2 indicates that the standard accurately reflects full length Akt (pSer473) content in samples.
      Linearity

      Table 5: Linearity of Dilution

      Lysate buffer was spiked with Akt (pSer473) and serially diluted in standard diluent buffer over the range of the assay. Linear regression analysis of sample values versus the expected concentration yielded a correlation coefficient of 0.99.
      Specificity

      Figure 3: Specificity

      The specificity of this assay for Akt phosphorylated at Ser473 was confirmed by peptide competition. The data presented in figure 3 shows that only the phosphopeptide containing the phosphorylated serine could block the ELISA signal. The same sequence containing non-phosphorylated serine at position 473 did not block the signal. The assay was found to have no cross-reactivity with the following recombinant phosphoproteins tested at 100 ng/ml: p38 MAPK, p44 ERK1, p42 ERK2, JNK1, human insulin receptor, rat insulin receptor, and human HGFR (c-met). Note that the assay is specific for Akt1 phosphorylated at Ser473 and does not cross-react with phosphorylated Akt3 (data not shown). It may cross-react with phosphorylated Akt2, but this has not been extensively tested.


      Figure 4: Specificity

      Figure 4 shows that treatment with wortmannin, a specific inhibitor of PI-3 Kinase, inhibits phosphorylation of Akt in Jurkat cells. This inhibition occurs in a dose-dependent manner as quantitated by the Akt (pSer473) ELISA. Jurkat cells were treated with wortmannin at varying concentrations from 0-500 nM for 3 h, lysed and assayed in parallel for both Akt (Total) and Akt (pSer473). The amount of total Akt remained comparable, while the levels of phosphorylation at serine residue 473 decreased with increasing doses of wortmannin.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Triton® is a registered trademark of Rohm and Haas company
      InteractivePathways™ are trademark of EMD Chemicals, Inc.

      Productos y aplicaciones relacionados

      Suggested Product

      Número de referencia Descripción  
      71296 PhosphoSafe™ Extraction Reagent Precios y disponibilidad

      Productos relacionados por: Brand Facete

      PhosphoDetect™

      Categorías

      Life Science Research > Antibodies and Assays > Immunoassays > Enzyme-linked Immunosorbent Assay (ELISA) > Complete ELISA Kits
      Life Science Research > Protein Detection and Quantification > Immunoassays > Enzyme-linked Immunosorbent Assay (ELISA) > Complete ELISA Kits