CBA101 PI 3-K Assay Kit

CBA101
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      Overview

      Replacement Information

      Key Specifications Table

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      CBA101-1KIT
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          Description
          OverviewA sensitive and robust homogeneous PI 3-K assay that does not require radioactive materials or secondary (detector) enzymes or antibodies. The kit is ideally suited for automated screening (HTS) and can be read on any fluorimeter.
          Catalogue NumberCBA101
          Brand Family Calbiochem®
          SynonymsPhosphoinositide 3 Kinase Assay Kit
          Application Data


          Varying concentrations of enzyme were mixed with 1 µM substrate in cAB containing 75 µM ATP. Relative fluorescence units (RFU) decrease as a function of enzyme activity.

          Varying amounts of ATP were used in the absence or presence of enzyme. Percent activity was calculated based on the ΔRFU between the samples.

          Varying amounts of Wortmannin in 0.05% DMSO were added to enzyme (32 nM) and substrate (1 µM) in cAB containing 20 µM ATP.

          Statistical data were produced using 0% (top) and 100% phospho-calibrators (bottom) or 27 nM PI 3-Kα (middle). Z′-factors of 0.8 and 0.7 were obtained using phospho-calibrators or reacted samples, respectively. A Z′-factor of >0.5 indicates a robust assay.
          Materials Required but Not Delivered PI 3-Kα,β,γ, or δ (purified enzyme; e.g., Cat. No. 526555, 526554, 526558, 526556)
          DTT Cat. No. 233155)
          Substrate (BODIPY-TMR-PtdIns)
          Calibrator (BODIPY-TMR-PtdIns(3)P)
          ATP (Cat. No. 1191)
          Wortmannin (Cat. No. 681675; optional control inhibitor for inhibitor assays)
          LY-294002 (Cat. No. 440204; optional control inhibitor for inhibitor assays)
          96-well plate (black)
          Fluorescent Microplate Reader
          References
          ReferencesRuckle, T., et al. 2006. Nat. Rev. Drug Discov. 11, 903.
          Zhao, J.J., et al. 2006. Proc. Natl. Acad. Sci. USA 103, 16296.
          Zhao, J.J. and T.M Roberts. 2006. Sci STKE 365, 52.
          Zhao, J.J., et al. 2005. Proc. Natl. Acad. Sci. USA 102, 18443.
          Meier T.I., et al. 2004. Protein Expr. Purif. 35.218.
          Product Information
          Form100 Tests
          Format96-well plate
          Kit containsAssay Buffer, Coordination Buffer, Sensor Stock, Sensor Dilution Buffer, Trivalent Metal Ion, Post Reaction Buffer, and a user protocol.
          Applications
          Biological Information
          Assay time2 h
          Sample TypePurified enzyme
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 10-23/24/25-39/23/24/25

          Flammable.
          Toxic by inhalation, in contact with skin and if swallowed.
          Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.
          S PhraseS: 1/2-36/37-45-63-A09

          Keep locked up and out of the reach of children.
          Wear suitable protective clothing and gloves.
          In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
          In case of accident by inhalation: remove casualty to fresh air and keep at rest.
          Product Usage Statements
          Intended useThe Calbiochem® PI 3-K Assay Kit is a homogeneous assay designed to measure PI 3-K activity using purified enzyme preparations.
          Storage and Shipping Information
          Ship Code Blue 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 +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 containsAssay Buffer, Coordination Buffer, Sensor Stock, Sensor Dilution Buffer, Trivalent Metal Ion, Post Reaction Buffer, and a user protocol.
          Specifications

          Documentation

          PI 3-K Assay Kit Certificates of Analysis

          TitleLot Number
          CBA101

          References

          Reference overview
          Ruckle, T., et al. 2006. Nat. Rev. Drug Discov. 11, 903.
          Zhao, J.J., et al. 2006. Proc. Natl. Acad. Sci. USA 103, 16296.
          Zhao, J.J. and T.M Roberts. 2006. Sci STKE 365, 52.
          Zhao, J.J., et al. 2005. Proc. Natl. Acad. Sci. USA 102, 18443.
          Meier T.I., et al. 2004. Protein Expr. Purif. 35.218.
          User Protocol

          Revision09-July-2012 JSW
          SynonymsPhosphoinositide 3 Kinase Assay Kit
          Form100 Tests
          Format96-well plate
          StorageUpon arrival store the entire contents of the kit at 4°C.
          Intended useThe Calbiochem® PI 3-K Assay Kit is a homogeneous assay designed to measure PI 3-K activity using purified enzyme preparations.
          Principles of the assayThe Calbiochem® PI 3-K Assay Kit is ideally suited for automated screening of PI 3-K activity and can be read on any fluorimeter. In the figure below, the dye-labeled [red starburst] substrate (a) becomes phosphorylated by PI3-K in the presence of ATP (b). The provided sensor [purple star], which is a proprietary trivalent metal ion, associates with the dye-labeled, phosphorylated substrate (c). Fluorescence intensity becomes quenched as each sensor associates with phosphorylated moieties on the substrate. Fluorescence intensity decreases in direct proportion to the amount of phosphorylated substrate.

          Figure 1: Assay Principle

          Materials provided• Assay Buffer 3 (Kit Component No. JA9541-10ML): 1 vial, 10 ml, supplied as 50 mM MgCl₂, 25 mM HEPES, 0.05% sodium azide, pH 7.0
          • Coordination Buffer (Kit Component No. JA9542-600UL): 1 vial, 600 µl, supplied as 1X, pH 9.5
          • Sensor Stock (Kit Component No. JA9543-45UL): 1 vial, 45 µl, supplied in DMSO/methanol (1:1)
          • Sensor Dilution Buffer (Kit Component No. JA9544-10ML): 1 vial, 10 ml, supplied as a MES/NaCl-based buffer, 0.05% sodium azide
          • Trivalent Metal Ion (Kit Component No. JA9554-10UL): 1 vial, 10 µl, supplied as 50 mM in 0.5 N HCl
          • Post Reaction Buffer (Kit Component No. JA9555-1EA): 1 vial (lot-specific volume), supplied as NaCl-based buffer, 0.05% NaN₃
          Materials Required but not provided PI 3-Kα,β,γ, or δ (purified enzyme; e.g., Cat. No. 526555, 526554, 526558, 526556)
          DTT Cat. No. 233155)
          Substrate (BODIPY-TMR-PtdIns)
          Calibrator (BODIPY-TMR-PtdIns(3)P)
          ATP (Cat. No. 1191)
          Wortmannin (Cat. No. 681675; optional control inhibitor for inhibitor assays)
          LY-294002 (Cat. No. 440204; optional control inhibitor for inhibitor assays)
          96-well plate (black)
          Fluorescent Microplate Reader
          Precautions and recommendations• Compatible Substances: To determine the tolerance of the Sensor to substances commonly used for screening, the various substances were added to samples containing either 0% or 100% of control concentration of phospholipid in complete Assay Buffer (cAB). Following addition of Sensor, the S/B and the ΔRFU between the lipid controls were determined. Compatible substance concentrations listed are those that resulted in <15% loss of ΔRFU and <15% loss of S/B.

          Table 1: Compatible Substances


          • Working with Lipids: Due to their hydrophobic nature, lipids tend to adhere to plastic surfaces and can be challenging to work with. The following recommendations may help in working with these substrates:
          • Lyophilized lipids can be dissolved in 50 mM HEPES, pH 7.4 to achieve a stock concentration of 200 µM. To avoid freeze/thaw cycles, the stock should be dispensed into aliquots and stored at -70°C. For long-term storage, lipids can be dissolved in methanol instead of HEPES, dispensed into aliquots, and stored at -70°C.
          • Use siliconized tips and tubes to reduce loss of lipids.
          • Sonicate working solution of lipids before dispensing into wells.
          • Phosphatidylinositol performs best when prepared 10-30 min prior to use.
          • Terminating the Enzyme Reaction: Addition of Post Reaction Buffer will terminate the enzyme activity of up to 100 nM PI 3-Kα. If higher concentrations of enzyme are used it may be necessary to supplement the Post Reaction Buffer with EDTA. EDTA can be added fresh, directly to the Post Reaction Buffer, before addition to the wells, using the following guidelines:
          • Add a volume of concentrated EDTA to Post Reaction Buffer to obtain a 20 mM working solution. For example, add 80 µl of a 250 mM EDTA stock solution to 920 µl Post Reaction Buffer to obtain a 20 mM working solution of Post Reaction Buffer/EDTA. Following addition of 10 µl of this working solution to the wells (see Detailed Protocol), the final concentration of EDTA is 5 mM; do not exceed 10 mM EDTA per well, final concentration.
          • EDTA may precipitate from solution when stored at 4°C. If large volumes of Post Reaction Buffer containing EDTA are prepared, store the working solution at room temperature to avoid precipitation.
          Reagent preparation• 20X Sensor: Add 37 µl Sensor Stock to 458 µl Coordination Buffer. Add 5 µl Trivalent Metal Ion. Incubate for 1 h at room temperature. Protect from light. 20X Sensor is stable for up to 4 weeks at 4°C. • Complete Assay Buffer (cAB): add DTT to Assay Buffer to obtain a 5 mM final concentration of DTT. Note: Equilibrate to room temperature and use within 8 h of preparation • 3X Substrate (1 µM final concentration): Prepare enough 3X Substrate for the number of assays to be performed; 10 µl per well is required. Dilute Substrate in cAB to achieve a substrate working solution of 3 µM. • 6X ATP Solution (100 µM final concentration): Prepare enough 6X ATP for the number of assays to be performed; 5 µl per well is required. Dilute ATP to in cAB to achieve a working ATP solution that is 600 µM. • 6X Inhibitor Solutions: Prepare enough 6X Inhibitor Solution for the number of assays to be performed; 5 µl per well is required. To the appropriate amount of cAB, add Inhibitors to achieve a concentration that is 6X the desired final concentration. If no inhibitor is used, adjust the volume with cAB. • 3X Enzyme Working Solution: Prepare enough 3X Enzyme Working Solution for the number of assays to be performed; 10 µl per well is required. To the appropriate amount of cAB, add Enzyme that is 3X the desired final concentration; 1-100 nM final concentration is recommended, depending on the application.
          Detailed protocolNote: To control for background fluoresence, prepare a control containing all components except substrate. the background RFU can be subtracted from sample RFU to reduce non-specific signal.
          1. Dispense the following assay components into designated wells of a black 96-well plate:
          • 5 µl 6X Inhibitor Solution
          • 5 µl 6X ATP solution
          • 10 µl 3X Substrate (or cAB for controls)
          • 10 µl 3X Enzyme Working Solution
          Total volume = 30 µl
          Cover the plate and incubate for 30-90 min.
          2. Add 10 µl Post Reaction Buffer; EDTA can be added fresh to stop the enzyme reaction (see Precautions and Recommendations).
          3. Prepare enough 1X Sensor for the number of assays to be performed; 80 µl well is required. Just prior to use, dilute 20X Sensor (above) 1:20 with Sensor Dilution Buffer. Add 80 µl 1X Sensor to each well. Cover the plate and incubate for 60 min at room temperature.
          4. Following incubation, shake the plate and monitor the fluorescence at an excitation wavelength of 540 nm and an emission wavelength of 580 nm. Plates may be read for up to 5 h after performing the assaying without loss of signal. Increasing the time of incubation with Sensor decreases the raw RFU, but the S/B remains constant.
          Example dataNote: Graphs were generated using GraphPad Prism™ Software. Curve fit was performed using sigmoidal dose response (variable slope). Error bars represent one standard deviation from the mean of two replicates.

          Figure 2: Enzyme Dose Response Curve

          Varying concentrations of enzyme were mixed with 1 µM substrate in cAB containing 75 µM ATP. Relative fluorescence units (RFU) decrease as a function of enzyme activity.

          Figure 3: ATP Tolerance Curve

          Varying amounts of ATP were used in the absence or presence of enzyme. Percent activity was calculated based on the ΔRFU between the samples.

          Figure 4: Inhibitor Curve

          Varying amounts of Wortmannin in 0.05% DMSO were added to enzyme (32 nM) and substrate (1 µM) in cAB containing 20 µM ATP.

          Figure 5: Statistics

          Statistical data were produced using 0% (top) and 100% phospho-calibrators (bottom) or 27 nM PI 3-Kα (middle). Z′-factors of 0.8 and 0.7 were obtained using phospho-calibrators or reacted samples, respectively. A Z′-factor of >0.5 indicates a robust assay.

          Registered TrademarksCalbiochem® is a registered trademark of , KGaA, Darmstadt, Germany.
          Interactive Pathways™ is a trademark of , KGaA, Darmstadt, Germany.
          GraphPad Prism™ is a trademark of GraphPad.