Overview

Kinases play a pivotal role in many signal transduction cascades and consequently kinase activities remain a key focus of academic and pharmaceutical research. SPA beads can be used in the study of tyrosine and serine/threonine kinases using both peptide and whole protein substrates. In the SPA assay, substrate is captured to an SPA bead. If the substrate has been phosphorylated, the beta energy from the radiolabeled phosphate group will be in close enough proximity to interact with the scintillant within the SPA bead, producing signal. Unreacted ³³P-gamma-ATP will not associate with the SPA bead, and will not be able to generate signal.

Figure 1. SPA Kinase Assay

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What do I need to run this assay?

• ³³P-gamma-ATP (Refer to table in next section.)
• Cold ATP, test compounds as needed
• Substrate-of-interest, tagged for SPA bead capture
• SPA bead
• Kinase
• White plates or tubes (If reading in microplate format, we recommend white OptiPlates™ or white-walled, clear bottom IsoPlates. See next section.)
• Liquid scintillation counter (such as a Tri-Carb® counter) or high-throughput detection instrument (MicroBeta® counter or TopCount® counter for scintillation beads; ViewLux™ detector for imaging beads) as appropriate

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Products and catalog numbers

Radiolabeled ATP

• ³³P-gamma-ATP is preferred over ³²P-gamma-ATP because of its lower energy, leading to lower assay background
• EasyTides products contain a dye in the buffer to aid with pipetting, and can be stored at 2-8°C
• NEG602K (high-throughput screening formulation) has a stabilizer in the buffer that keeps the product more stable at room temperature while setting up plates for high throughput screening.

SPA beads

SPA bead types

Plates

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Protocol-in-brief

Off-bead format: In off-bead assays, all the components are present in solution. The reaction continues in the absence of bead, is then terminated in the presence of bead capturing the radiolabelled products. This format is ideal for looking at enzyme kinetic studies and can also be compared to other formats. Steric hindrance (where the presence of bead during a reaction may interfere with enzyme/substrate action) is not an issue. However, as beads have a finite binding capacity per unit mass they must either be used in excess to ensure adequate capture, or known amounts of bead are used to capture constant amounts of product.

On-bead format: In this case the substrate is immobilized onto the bead surface and thus the reaction takes place in the presence of bead. This format allows real time monitoring of reactions, and good signals can be achieved with smaller amounts of substrate and bead. This format is not suitable for all enzymes, as there may be a large degree of steric hindrance leading to reduced substrate availability.

SPA kinase protocol-in-brief

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Assay development

Enzyme titration

• Enzyme source and purity: use several known controls to validate issues relating to enzyme performance.
• An enzyme titration experiment should be set up to determine how much enzyme to use in the final, optimized reaction.
• Investigate the necessity for enzyme pre-activation or of ancillary co-factors required (DAG, DNA) for full and specific activity.
• If the enzyme itself autophosphorylates, initial pre-incubation with cold ATP may be required.

Kinase titration. Erk1 kinase was assayed using biotinylated MBP as a substrate.

Substrate titration

• Consider peptide vs full-length protein issues with respect to Km and number of potential kinase sites. Ideally the Km should be as low as possible.
• Ensure the substrate tag used in capture (e.g. biotin, antibody, GST or His-tag) is far enough from phosphorylation sites to minimize interference in binding or phosphorylation.
• To determine an optimal amount of substrate to use, it is helpful to have some knowledge of the kinetic parameters of the kinase, such as Km, which can be used to help determine the optimal mass of substrate. In the absence of such information, a substrate titration should be set up, such that all other reactants are kept constant (including amount of bead).

Substrate titration. This figure shows the titration of biotinylated MBP substrate against enzyme concentration (Erk-1). The fall in counts for concentrations greater than 3.5 µM substrate shows that the bead binding capacity has been exceeded. At 0.5 µM substrate, the substrate very quickly becomes limiting. Reaction stopped in standard stop buffer, using 0.5 mg streptavidin-coated PVT beads per well in 96-well format.

Bead titration

• Typically use 0.5 mg of bead per well (in 96-well format) for streptavidin beads. Other bead types should be titrated to maximize capture of tagged component of assay.
• As with the substrate, you can set up a series of reactions varying only the amount of bead in a titration experiment. Common titrations will go up to 2.0 mg bead per well. Remember to subtract out background counts.

YSi bead titration using an ERK-1 reaction in 384-well plates. Substrate is biotinylated MBP, 50 pmoles per well. This figure shows that between 0.25 mg and 0.5 mg of bead is sufficent to capture all of the substrate, and hence all the product, in the reaction.

The kinase reaction

• Assay buffer
  Use known buffers suggested by literature if possible, making certain to include a phosphatase inhibitor (e.g. sodium orthovanadate at 100 µM). Note that the more cold ATP is used, the lower the potential signal, but there will be less of a bias towards competitive inhibitors of the ATP binding site.
  
  Alternative assay buffers:
  A) 50 mM Tris-HCl, pH 8.0, 1 mM DTT, 100 µM sodium orthovanadate, 10 mM MgCl2, 1 µM ATP, 0.75 µM biotinylated peptide substrate, 0.2-0.5 µCi gamma-³³P-ATP
  B) 50 mM MOPS, pH 7.2, 1 µM ATP, 5 mM MgCl₂, 2.5 µM biotinylated protein substrate, 0.2-0.5 µCi gamma-³³P-ATP

• Keep the reaction volume low (no more than 50 µL in a 96-well plate) to accommodate a larger stop reagent volume (see below).
• Incubation period should commonly be in the 30-60 minute range. If uncertain, a kinase time course analysis should be performed to ascertain that the signal is not compromised by increased NSB or noise in the assay.

Stop reaction

• For streptavidin-coated PVT beads: 50 µM ATP, 5 mM EDTA, 0.1% (v/v) Triton X-100 in PBS (without calcium or magnesium)
• For streptavidin-coated YSi beads: 50 mM ATP in PBS (without calcium or magnesium)
• For PVT beads use a PBS-based stop reagent containing 5 mM EDTA, 50 µM ATP and 0.1% Triton X-100.
• For PVT copper chelate beads: 50 mM ATP, 0.5% (w/v) BSA, 1% (v/v) Triton X-100 in PBS (without calcium or magnesium)
• For YSi beads, use 100 µM ATP, omit EDTA, and raise pH to 11.0.
• For YSi copper chelate beads: 50 mM ATP, 0.2% (w/v) BSA, 1% (v/v) Triton X-100 in PBS (without calcium or magnesium)

Assay miniaturization

• SPA miniaturization can be achieved by:

  • Use of 96-well concentrations in a reduced volume
  • Use of 96-well mass of reagent in a reduced volume
  • Full optimization in 384-well format

Figure. 96-well vs. 384-well assay, full optimization

• Overall, we try to set as large a final assay volume as possible to avoid non-proximity effects. For 384-well microplates, this equates to a 75 µL volume, following addition of bead/stop reagent.

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Tips and FAQs

Q. I have to use a large amount of peptide substrate in my assay - too much for a viable amount of bead to capture. What can I do?
A. While it is always desirable to be able to capture all of the input substrate on the amount of bead available, it is not always possible. There are two ways to account for this:

1. Calculate the proporation of substrate that is captured by the mass of bead used, then multiply the final SPA signal by this proportion
2. Remove an aliquot from the stopped reaction that only contains the mass of substrate that can be captured on your required bead mass. This again gives a proportion of the total signal in the assay.

Q. Do I have to stop my kinase assays?
A. All SPA enzyme assays need to be stopped to prevent drift. For kinase assays there are a number of different stop reagents that are effective. Refer to the Assay Optimization section above for more details.

Q. Once I have stopped my reaction, can I count it?
A. No. The use of ³³P in SPA brings with it the requirement to pack the beads, both to reduce background caused by non-proximity effect and to increase the harvesting of the specific counts.

Q. How do I pack my beads?
A. There are three options for bead packing: gravity settling, centrifugation, and CsCl flotation. The latter two are only appropriate for PVT-based beads, as the dense YSi beads settle extremely rapidly under gravity and are too heavy to float.

For microplates, centrifugation at 1,000 x g for 10 minutes is sufficient. You must ensure that the plates are properly balanced in the rotor. We have found swing out rotors to be effective in this application. For floation a final concentration of 2-3 M CsCl is required in the well. This will lead to maximum signal being reached after approximately 90 minutes, as opposed to 7+ hours with gravity for PVT beads. This signal is stable for at least 24 hours.

Q. Can I mix the CsCl solution with my stop reagent, to reduce the number of pipetting steps?
A. Yes, CsCl will have no effect on streptavidin-biotin binding, however, it may not be compatible with the copper chelate or glutathion beads. Remember that the beads will float in this solution, so you must keep the bead suspension homogenous by stirring to ensure accurate sampling.

Q. Should I try to keep the volume as high as possible in the well?
A. Yes, using as large a final volume as possible in the well helps to reduce the non-proximity background. We routinely use a 250 μL final volume (50 μL assay volume, 200 uL stop/floatation mix) in a 96-well format.

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Posters and Guides

• SPA bead technology knowledge base page

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Citations

1. Lingaraj, T. et al. A High-Throughput Liposome Substrate Assay with Automated Lipid Extraction Process for PI 3-Kinase. Journal of Biomolecular Screening 13, 906 -911 (2008). Link
2. Soriano, A. et al. Escherichia coli acetyl-coenzyme A carboxylase: characterization and development of a high-throughput assay. Anal. Biochem 349, 268-276 (2006). Link
3. Van Aller, G.S. et al. Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay. Anal. Biochem 383, 311-315 (2008). Link

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Other 爱游戏平台注册登录 kinase technologies

• LANCE Ultra TR-FRET kinase assays
• LANCE Classic TR-FRET kinase assays
• AlphaScreen SureFire no-wash cellular kinase assays
• AlphaScreen/AlphaLISA kinase assays
• DELFIA fluorescence kinase assays

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Custom services at 爱游戏平台注册登录

爱游戏平台注册登录 offers custom radiochemicals (including GMP-certified radiochemicals), custom SPA beads, custom plate barcoding, and custom assay development. If you are interested in custom services, please contact our custom teams:

ON>POINT® Custom Services

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