Millipore Sigma Vibrant Logo
Attention: We have moved. Merck Millipore products are no longer available for purchase on MerckMillipore.com.Learn More

CBA102 Sphingosine Kinase Assay Kit

Sphingosine Kinase Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: SPK1 Assay Kit

CBA102
  
Purchase on Sigma-Aldrich

Overview

Replacement Information
Description
Overview

This product has been discontinued.





A sensitive and robust homogeneous sphingosine kinase 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 NumberCBA102
Brand Family Calbiochem®
SynonymsSPK1 Assay Kit
Application Data

Varying concentrations of enzyme were mixed with 1 µM substrate in cAB containing 100 µM ATP. Relative fluorescence units (RFU) decrease as a function of zyme activity.

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

Varying amounts of DESPH in 1% DMSO were added to enzyme (30 nM) and substrate (1 µM) in cAB containing 50 µM ATP.

Statistical data were produced using 0% (top) 100% phospho-calibrators (bottom) or 30 nM sphingosine kinase (middle). Z′-factors of 0.9 and 0.6 were obtained using phospho-calibrators or reacted samples, respectively. A Z′-factor of >0.5 indicates a robust assay.
Materials Required but Not Delivered• Sphingosine Kinase (purified enzyme)
• DTT (Cat. No. 233155)
• ATP (Cat. No. 1191)
• EDTA(Cat. No. 34103)
• 96-well plate (black)
• Fluoresence Microplate Reader
• TMR Sphingosine
• DESPH
References
ReferencesSpiegel, S, and Milstein, S. 2007. J. Biol. Chem. 282, 2125.
Taha, T.A.., et al. 2006. J. Biochem. Mol. Biol. 39, 113.
Taha, T.A., et al. 2005. FASEB J. 20, 482
Delon, C., et al. 2004. J. Biol. Chem. 279, 44763.
Ansari, M.A., et al. 2004. Brit. J. Haemat. 126, 758;
Armstrong, S.A., et al. 2004. Blood 103, 3544;
Abu-Duhier, F.M., et al. 2000. Brit. J. Haemat. 111, 3544.
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: 16-36/37-45-7-A09

Keep away from sources of ignition - No Smoking.
Wear suitable protective clothing and gloves.
In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Keep container tightly closed.
Product Usage Statements
Intended useThe Calbiochem® Sphingosine Kinase Assay Kit is a homogeneous assay designed to measure sphingosine kinase 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
Global Trade Item Number
Catalogue Number GTIN
CBA102 0

Documentation

Sphingosine Kinase Assay Kit Certificates of Analysis

TitleLot Number
CBA102

References

Reference overview
Spiegel, S, and Milstein, S. 2007. J. Biol. Chem. 282, 2125.
Taha, T.A.., et al. 2006. J. Biochem. Mol. Biol. 39, 113.
Taha, T.A., et al. 2005. FASEB J. 20, 482
Delon, C., et al. 2004. J. Biol. Chem. 279, 44763.
Ansari, M.A., et al. 2004. Brit. J. Haemat. 126, 758;
Armstrong, S.A., et al. 2004. Blood 103, 3544;
Abu-Duhier, F.M., et al. 2000. Brit. J. Haemat. 111, 3544.
User Protocol

Revision11-June-2009 JLN
SynonymsSPK1 Assay Kit
Form100 Tests
Format96-well plate
StorageUpon arrival store the entire contents of the kit at 4°C.
Intended useThe Calbiochem® Sphingosine Kinase Assay Kit is a homogeneous assay designed to measure sphingosine kinase activity using purified enzyme preparations.
Principles of the assayThe Calbiochem® Sphingosine Kinase Assay Kit is ideally suited for automated screening of sphingosine kinase activity and can be read on any fluorimeter. In the figure below, the dye-labeled [red starburst] substrate (a) becomes phosphorylated by sphingosine kinase 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 1 (1X) (Kit Component No. JA9545-10ML): 1 vial, 10 ml, supplied as 10 mM Tris, 10 mM MgCl₂, 0.015% Triton®X-100 detergent, 0.05% sodium azide, pH 7.2
• Coordination Buffer (Kit Component No. JA9546-600UL): 1 vial, 600 µl, supplied as 1X, pH 9.5
• Sensor Stock (Kit Component No. JA9547-22UL): 1 vial, 22 µl, supplied in DMSO/methanol (1:1)
• Sensor Dilution Buffer (Kit Component No. JA9548-10ML): 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 0.1 N HCl
• Post Reaction Buffer (Kit Component No. JA9555-1.5ML): 1 vial, 1.5 ml, supplied as NaCl-based buffer, 0.05% NaN₃
Materials Required but not provided• Sphingosine Kinase (purified enzyme)
• DTT (Cat. No. 233155)
• ATP (Cat. No. 1191)
• EDTA(Cat. No. 34103)
• 96-well plate (black)
• Fluoresence Microplate Reader
• TMR Sphingosine
• DESPH
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


• Terminating the Enzyme Reaction: To terminate the enzymatic activity, EDTA can be added directly to the Post Reaction Buffer before addition to the wells using the following recommendations:
• Add a volume of concentrated EDTA solution to Post Reaction Buffer to obtain a 20 mM working solution. For example, a 20 mM solution is obtained by addition of 80 µl of a 250 mM EDTA solution to 920 µl Post Reaction Buffer. Upon addition of 10 µl of this solution to wells, a final concentration of 5 mM in the well is obtained. Do not exceed 10 mM EDTA/well (final concentration).
• EDTA may precipitate from solution at 4°C. If large volumes of Post Reaction Buffer containing EDTA are prepared, store the solution at room temperature to avoid precipitation.
Reagent preparation• 50X Sensor: Add 15 µl Sensor Stock to 183 µl Coordination Buffer. Add 2 µl Trivalent Metal Ion. Incubate for 1 h at room temperature. Protect from light • 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; 2-200 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 50X Sensor (above) 1:50 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 100 µM ATP. Relative fluorescence units (RFU) decrease as a function of zyme 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 samples.

Figure 4: Inhibitor Curve

Varying amounts of DESPH in 1% DMSO were added to enzyme (30 nM) and substrate (1 µM) in cAB containing 50 µM ATP.

Figure 5: Statistics

Statistical data were produced using 0% (top) 100% phospho-calibrators (bottom) or 30 nM sphingosine kinase (middle). Z′-factors of 0.9 and 0.6 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 EMD Chemicals, Inc.
Triton® is a registered trademark of Dow Chemical Commpany.
Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
GraphPad Prism™ is a trademark of GraphPad.