CBA019 K-LISA™ Akt Activity Kit

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CBA019
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Key Specifications Table

Detection Methods
Colorimetric

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CBA019-1KIT
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      The K-LISA™ Akt Activity Kit is a rapid, sensitive, ELISA-based activity assay suitable for measuring the kinase activity of purified or partially purified Akt preparations, in vitro Akt inhibitor screening, and for assessing the regulation of Akt in cell signaling. The assay utilizes a biotinylated peptide substrate (GRPRTSSFAEG) that is phosphorylated on the second serine by Akt1, Akt2, Akt3, SGK (Serum Glucocorticoid Kinase), and MSK1. Biotinylated Akt substrate and sample containing Akt are incubated in the presence of ATP in wells of a streptavidin-coated 96-well plate, which allows for phosphorylation and substrate capture in a single step.

      Catalogue NumberCBA019
      Brand Family Calbiochem®
      Application Data
      Near-confluent MCF-7 cells were stimulated with IGF-1 (100 ng/ml) or IGF-1 (100 ng/ml) and 17β-estradiol (500 nM) for 30 min at 37°C in a CO2 incubator. For inhibition of Akt, cells were first pre-incubated at 37°C in a CO2 incubator for 15 min in the presence of cell-permeable Akt Inhibitor II (Cat. No. 124008) followed by stimulation with IGF-1 (100 ng/ml) for 30 min at 37°C. Cell lysates were prepared using 0.5 ml PhosphoSafe™ Extraction Buffer per 10 cm cell culture dish, per the standard protocol. Cell lysates were either used immediately or stored at -70°C until use. Equal amounts of total protein (1.5 mg) were immunoprecipitated and the activity was determined as outlined in protocol B under the Detailed Protocol section.
      Materials Required but Not Delivered Akt-containing sample
      Purified Akt (optional positive control; e.g., Akt, HisTag®, Activated, Human, Recombinant, S. frugiperda, Cat. No. 14-276)
      Anti-Akt (for immunoprecipitating active Akt from crude cell lysates) (Cat. No. ST1088)
      Protein A Agarose Suspension (Cat. No. IP02), Protein G-Plus Agarose Suspension (Cat. No. IP04), or Protein A/Protein G-Plus Agarose Suspension (Cat. No. IP05) (for immunoprecipitation of Akt)
      1X PBS
      Wash bottle or multichannel dispenser for washing
      Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably in the dual wavelength mode at 450 nm against a reference wavelength of 540 or 595 nm.
      References
      ReferencesLiau, S.S., et al. 2006. J. Gastrointest. Surg. 10, 1254.
      Hill, M.M. et al. 2002. Pharmacol. Ther. 93, 243.
      Shin, I. et al. 2002. Nat. Med. 8, 1136.
      Zheng, et al. 2002. J. Biol. Chem. 42, 39379.
      Brazil, D.P. et al. 2001. Trends Biochem. Sci. 26, 657.
      Medema, R.H., et al. 2000. Nature 404, 782.
      Datta, S.R., et al. 1999. Genes Dev. 13, 2905.
      Franke, F.T., et al. 1997. Cell 88, 435.
      Alessi, D.R., et al. 1996. EMBO J. 15, 6541.
      Product Information
      Unit of DefinitionOne unit is defined to the amount of enzyme required to transfer 1 nmol phosphate per min to substrate at 30° C.
      Biotinylated Akt substrate, phosphoserine detector antibody, streptavidin coated 96-well plate strips, HRP-conjugated secondary antobody, serine/threonine kinase assay buffer, ATP/MgCl₂, inhibitor, wash buffer, TMB substrate, stop solution, EDTA solution, plate sealers, and a user protocol. Purified Akt is available separately (Cat. No. 124006).
      Detection methodColorimetric
      Declaration*Sold under license of U.S. patent 6,441,140 Manufactured by Cell Signaling
      Form96 Tests
      Format96-well plate
      Kit containsStreptavidin-Coated 96-Well Plate, Akt Substrate, Biotinylated, Phosphoserine Detection Antibody, Antibody Diluent, Kinase Assay Buffer, 5X ATP/MgCl₂ Mix, 100X Inhibitor, Kinase Stop Solution, ELISA 20X Plate Wash Concentrate, HRP-Antibody Conjugate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol. Sold under license of U.S. patent 6,441,140. Manufactured by Cell Signaling.
      Applications
      Biological Information
      Assay range6-100 mU/well
      Assay time3 h
      Sample TypeCell lysates, tissue extracts, purified enzyme
      Physicochemical Information
      Sensitivity0.05 mUnits
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe K-LISA™ Akt Activity Kit is suitable for measuring the kinase activity of purified or partially purified Akt preparations, in vitro Akt inhibitor screening, and for assessing the regulation of Akt in cell signaling.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage -20°C
      Storage ConditionsUpon arrival store the Streptavidin-Coated 96-Well Plate at 4°C and the remaining components of the kit at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsStreptavidin-Coated 96-Well Plate, Akt Substrate, Biotinylated, Phosphoserine Detection Antibody, Antibody Diluent, Kinase Assay Buffer, 5X ATP/MgCl₂ Mix, 100X Inhibitor, Kinase Stop Solution, ELISA 20X Plate Wash Concentrate, HRP-Antibody Conjugate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol. Sold under license of U.S. patent 6,441,140. Manufactured by Cell Signaling.
      Specifications

      Documentation

      K-LISA™ Akt Activity Kit Certificates of Analysis

      TitleLot Number
      CBA019

      References

      Reference overview
      Liau, S.S., et al. 2006. J. Gastrointest. Surg. 10, 1254.
      Hill, M.M. et al. 2002. Pharmacol. Ther. 93, 243.
      Shin, I. et al. 2002. Nat. Med. 8, 1136.
      Zheng, et al. 2002. J. Biol. Chem. 42, 39379.
      Brazil, D.P. et al. 2001. Trends Biochem. Sci. 26, 657.
      Medema, R.H., et al. 2000. Nature 404, 782.
      Datta, S.R., et al. 1999. Genes Dev. 13, 2905.
      Franke, F.T., et al. 1997. Cell 88, 435.
      Alessi, D.R., et al. 1996. EMBO J. 15, 6541.
      User Protocol

      Revision23-August-2016 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      StorageUpon arrival store the Streptavidin-Coated 96-Well Plate at 4°C and the remaining components of the kit at -20°C.
      Intended useThe K-LISA™ Akt Activity Kit is suitable for measuring the kinase activity of purified or partially purified Akt preparations, in vitro Akt inhibitor screening, and for assessing the regulation of Akt in cell signaling.
      BackgroundAkt, also known as protein kinase B, is a serine/threonine kinase activated in response to growth factors, cytokines, and other diverse stimuli. It plays a role in the regulation of many cellular processes such as cell-cycle progression, apoptosis, and glucose uptake. Over-expression or constitutive activation of Akt is associated with many types of cancer. There are three Akt isoforms, each of which consists of a conserved domain structure: an amino terminal pleckstrin homology (PH) domain, a central kinase domain, and a carboxy-terminal regulatory domain that contains a hydrophobic motif. The PH domain plays a significant role in recognition of Akt by upstream kinases, as well as membrane translocation. Phosphorylation of Thr308 in the central kinase domain and Ser473 in the hydrophobic motif is necessary for full activation. Akt provides a survival signal that protects cells from apoptosis induced by various stresses. These signals may promote survival through downstream targets such as, Bad, GSK-3, IκB kinase β (IKK-β), pro-caspase-9, or members of the Forkhead family of transcription factors, FKHRL1 and AFX. Akt may also promote cell proliferation by phosphorylation of Raf protein kinase. The recent discovery that the tumor suppressor, PTEN (phosphatase and tensin homolog), is a cellular inhibitor of Akt, suggests that Akt is an important factor in oncogenesis. Inhibition of the Akt oncogenic pathway could lead to apoptosis of cancer cells that express constitutively active Akt. Thus, specific inhibitors of Akt are of interest in clinical research.
      Principles of the assayThe K-LISA™ Akt Activity Kit is an ELISA-based activity assay that utilizes a biotinylated peptide substrate (GRPRTSSFAEG) that is phosphorylated on the second serine by Akt1, Akt2, Akt3, SGK (Serum Glucocorticoid Kinase), and MSK1. Biotinylated Akt Substrate and sample containing Akt are incubated in the presence of ATP in wells of the Streptavidin-Coated 96-Well Plate, which allows for phosphorylation and substrate capture in a single step. The phosphorylated substrate is detected with the Phosphoserine Detection Antibody, followed by the HRP-Antibody Conjugate and color development with TMB Substrate. Sensitivity is increased by the addition of ELISA Stop Solution, and relative activity is determined by reading the absorbance at dual wavelengths 450/540 nm or 450/595 nm. Recommended sample types include Akt that has been immunoprecipitated from crude cell lysates or purified Akt protein. Addition of Inhibitor (staurosporine) serves as a negative control. Inhibition profiles can be generated based on Akt activity in the presence and absence of test inhibitor(s).
      Materials provided• Streptavidin-Coated 96-Well Plate (Kit Component No. JA7912): supplied as 8-well strips, pre- blocked
      • Akt Substrate, Biotinylated (Kit Component No. JA7910): supplied at 1 mg/ml
      • Phosphoserine Detection Antibody (Kit Component No. JA7911): supplied at 1 mg/ml
      • Antibody Diluent (Kit Component No. JA7917):
      • Kinase Assay Buffer (Kit Component No. JA7913): supplied as a 5X solution
      • 5X ATP/MgCl2 Mix (Kit Component No. JA7914)
      • 100X Inhibitor (Kit Component No. JA7915): supplied as a 100 µM in DMSO
      • Kinase Stop Solution (Kit Component No. KP31517)
      • ELISA 20X Plate Wash Concentrate (Kit Component No. JA1617)
      • HRP-Antibody Conjugate (Kit Component No. JA7922): supplied as a 200X solution
      • TMB Substrate (Kit Component No. JA1608): ready-to-use
      • ELISA Stop Solution (Kit Component No. JA1616): 2.5N H₂SO₄
      • Plate Sealers (Kit Component No. JB155)
      Materials Required but not provided Akt-containing sample
      Purified Akt (optional positive control; e.g., Akt, HisTag®, Activated, Human, Recombinant, S. frugiperda, Cat. No. 14-276)
      Anti-Akt (for immunoprecipitating active Akt from crude cell lysates) (Cat. No. ST1088)
      Protein A Agarose Suspension (Cat. No. IP02), Protein G-Plus Agarose Suspension (Cat. No. IP04), or Protein A/Protein G-Plus Agarose Suspension (Cat. No. IP05) (for immunoprecipitation of Akt)
      1X PBS
      Wash bottle or multichannel dispenser for washing
      Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably in the dual wavelength mode at 450 nm against a reference wavelength of 540 or 595 nm.
      Reagent preparationPrepare all reagents immediately prior to use. The following table provides reagent preparation instructions to obtain the Volume of Working Solutions (WS) required for one 8-well strip:

      Table 1: Preparation of Reagants

      Detailed protocolA. Akt Kinase Activity and Inhibitor Screening Assay: recommended for purified or partially purified Akt preparations and for inhibitor screening.

      1. Remove required number of strips from the Streptavidin-Coated 96-Well Plate and place them in a 96-well frame. Return the unused strips to the foil pouch and reseal the entire edge of the pouch with tape. Store unused strips at 4°C.
      2. Add the following components to each well in the specified order:

      Table 2: Protocol

      *For inhibitor screening assay, if no inhibitor is to be included, up to 20 µl Akt Sample can be added or inhibitor can be substituted with 10 µl dH2O.


      3. Cover the plate with the Plate Sealer, mix with a plate shaker or equivalent, and incubate for 30 min at 30°C.
      4. Stop the kinase reaction by adding 10 µl Kinase Stop Solution to each well. (Reaction may also be stopped simply by discarding the contents of the wells).
      5. Discard the contents of the wells by shaking the plate over the sink and wash the Streptavidin-Coated 96-Well Plate 3-4 times with 1X ELISA Plate Wash, 200 µl/well. Shake out the wash solution and dry the wells by tapping the inverted plate on paper towels.
      6. Add 100 µl Phosphoserine Detection Antibody WS to each well and incubate for 1 h at room temperature.
      7. Wash the plate as indicated in step 5.
      8. Add 100 µl HRP-Antibody Conjugate WS to each well and incubate for 1 h at room temperature.
      9. Wash the plate as indicated in step 5.
      10. Add 100 µl TMB Substrate to each well and incubate for 5-20 min at room temperature.
      11. Add 100 µl ELISA Stop Solution to each well and read the absorbance at 450 nm, preferably with a reference wavelength set at 540-595 nm.

      B. Assay for Activity of Immunoprecipitated Akt: recommended for analysis of Akt activity in samples immunoprecipitated from cell lysates. Note: K-LISA™ Akt Activity Kit does not include an antibody for immunoprecipitation of active Akt from cell lysates. It is important is to use an antibody that will immunoprecipitate the enzyme without affecting the catalytic activity of Akt.

      1. Prepare cell lysates using PhosphoSafe™ Extraction Buffer (Cat. No. 71296) or other cell lysis buffer of choice. Pre-clear 0.5 ml cell lysate (total protein concentration = 2-5 mg/ml) by adding 15 µl (settled bed volume) Protein G-Plus, Protein A, or Protein A/Protein G-Plus agarose beads and incubating 15 min at 4°C.
      2. Centrifuge at 4000 rpm for 5 min at 4°C in a microcentrifuge to pellet the agarose beads. Transfer the pre-cleared lysate to a fresh tube.
      3. Add 5-10 µl appropriate Akt antibody (Cat. No. ST1088) to the pre-cleared lysate and incubate for 30 min at room temperature on an orbital shaker. (It is crucial to use an antibody that does not interfere with the catalytic activity of Akt and is capable of efficiently immunoprecipitating Akt from crude biological samples).
      4. Add 50 µl (settled bed volume) Protein G-Plus, Protein A, or Protein A/Protein G-Plus agarose beads and incubate for 60-90 min at room temperature on an orbital shaker.
      5. Centrifuge at 4000 rpm for 5 min at 4°C in a microcentrifuge to pellet the agarose beads (now bound to immunoprecipitated Akt). Carefully remove the supernatant with a pipet tip and discard.
      6. Wash the agarose beads with 0.5 ml 1X PBS, centrifuge at 4000 rpm for 5 min. at 4°C to pellet the agarose beads, and discard the supernatant. Repeat for a total of 3 washes. Carefully remove excess liquid following the final wash so as not to disturb the loose pellet of agarose beads.
      7. Add the following components in the specified order to the agarose beads in each tube, gently mix to evenly suspend the beads, and incubate for 30 min at 30°C.

      Table 3: Protocol 7


      8. Add 10 µl Kinase Stop Solution to each tube, mix briefly by gently tapping the tube. DO NOT VORTEX.
      9. Centrifuge at 4000 rpm for 5 min at 4°C to pellet the agarose beads. Transfer the supernatant to a fresh tube. The supernatant should be used immediately or stored at -70°C until the assay can be completed.
      10. Remove the required number of strips from the Streptavidin-Coated 96-Well Plate and place them in a 96-well frame. Return the unused strips to the foil pouch and seal the entire edge of the pouch with tape. Store unused strips at 4°C.
      11. Add 10-50 µl supernatant from step 9 to designated wells. If less than 50 µl is used, add 1X PBS to a final volume of 50 µl. Incubate for 30-60 min at 30°C.
      12. Aspirate the contents of each well and wash the Streptavidin-Coated 96-Well Plate 3-4 times with 1X ELISA Plate Wash. Dry the wells by tapping the inverted plate on paper towels.
      13. Add 100 µl Phosphoserine Detection Antibody-WS to each well and incubate for 1 h at room temperature.
      14. Wash the plate as in step 12.
      15. Add 100 µl HRP-Antibody Conjugate-WS to each well and incubate for 1 h at room temperature.
      16. Wash the plate as in step 12.
      17. Add 100 µl TMB Substrate to each well and incubate for 5-20 min at room temperature.
      18. Add 100 µl ELISA Stop Solution to each well and read the absorbance at 450 nm, preferably with a reference wavelength set at 540-600 nm.
      Assay characteristics and examples

      Figure 1: Activity of Purified Akt in the Presence and Absence of Staurosporine

      The activity of a recombinant human Akt1 Kinase (15-400 ng) was determined using protocol A as indicated under the Detailed Protocol section. The final concentration of staurosporine was 1 µM. Assay range: 10-200 ng (580 Units/mg).


      Figure 2: Activity of Akt Immunoprecipitated From Cell Lysate

      Near-confluent MCF-7 cells were stimulated with IGF-1 (100 ng/ml) or IGF-1 (100 ng/ml) and 17β-estradiol (500 nM) for 30 min at 37°C in a CO2 incubator. For inhibition of Akt, cells were first pre-incubated at 37°C in a CO2 incubator for 15 min in the presence of cell-permeable Akt Inhibitor II (Cat. No. 124008) followed by stimulation with IGF-1 (100 ng/ml) for 30 min at 37°C. Cell lysates were prepared using 0.5 ml PhosphoSafe™ Extraction Buffer per 10 cm cell culture dish, per the standard protocol. Cell lysates were either used immediately or stored at -70°C until use. Equal amounts of total protein (1.5 mg) were immunoprecipitated and the activity was determined as outlined in protocol B under the Detailed Protocol section.


      Figure 3: Activity of Akt Before and After Immunoprecipitation

      The black bars represent Akt activity from unstimulated or IGF-1-stimulated MCF-7 cells (as indicated) without immunoprecipitation (cell lysates were prepared as indicated in Figure 2). The white bars represent activity of immunoprecipitated Akt from unstimulated and stimulated MCF-7 cells (as indicated). Activity was assessed as outlined in protocol B under the Detailed Protocol section. The immunoprecipitation step ensures enrichment of Akt and measurement of Akt-specific activity.

      Sensitivity0.05 mUnits
      Assay Range6-100 mU/well
      Registered TrademarksCalbiochem® and His•Tag® are registered trademarks of EMD Chemicals, Inc.
      K-LISA™ and PhosphoSafe™ are trademarks of EMD Chemicals, Inc.
      Non-interfering Protein Assay™ is a trademark of Geno Technology, Inc.
      Technology®, a registered trademark of Cell Signaling Technology, Inc.