Alpha SureFire No-wash Cellular Kinase Assays

Alpha SureFire Assay Principle

In an AlphaLISA SureFire Ultra assay, the Donor bead is coated with streptavidin to capture the biotinylated antibody while the Acceptor bead is coated with a proprietary CaptSure™ agent that immobilizes the other antibody which is labeled with a CaptSure tag. The emission wavelength of the AlphaLISA Acceptor beads is out of hemoglobin absorption spectrum, making it a better tool for working with tissue lysates.

Our AlphaLISA SureFire Ultra assay offers several advantages:

• Simplified protocols
• User-friendly reaction volumes
• High signal-to-background
• High sensitivity
• Compatible with the use of spike antibodies (i.e., studies using biotherapeutic antibodies, antibody blockers, etc.)
• Compatible with tissue sample analysis and PBMCs
• Optimized for biotin-rich sample analysis

Assay features

• Cell-based endpoint assay, demonstrated applications on recombinant proteins, cell lines, primary cells, stem cells, tissues, plasma, and blood
• Homogeneous (no wash) assay - mix components in well and read in microplate based assay format (96-, 384-, and 1536-well amenable)
• Well suited for target identification through high-throughput screening and preclinical studies

Formats

The AlphaLISA SureFire assay kits are provided in the following formats:

What do I need to run this assay?

Available from Revvity:

AlphaLISA SureFire Ultra Assay Kits

Each kit includes:

• Validated antibodies specific to the target analyte (pre-mixed in Reaction Buffers)
• Streptavidin-coated Donor beads and CaptSure™-tagged Acceptor beads
• Lysis Buffer (5X), Activation Buffer, and Dilution Buffer
• Lyophilized positive control lysates
• Detailed assay protocol and reagent preparation guidelines

Recommended accessories:

• TopSeal™-A Adhesive Plate Seal – for secure sealing during incubation steps
• Microplates – optimized for assay performance and signal detection

Plate recommendations

For 2-Plate protocols (Adherent cells):

• Cell culture plate: Use a 96- or 384-well TC-treated plate (e.g., Revvity CulturPlate™ or ViewPlate™) for cell growth and treatment.
• Detection plate: Transfer lysates to a 384-well white assay plate such as:
  • OptiPlate™-384, White Opaque (Part #6007290)
  • AlphaPlate™-384, Light Gray (Part #6005350) – ideal for minimizing cross-talk

For 1-Plate protocols (Adherent cells):

• Use a 384-well TC-treated plate (e.g., CulturPlate™-384, Part #6007680) for cell culture, treatment, lysis, and detection in the same plate.

For suspension cells:

• Use either:
  • OptiPlate™-384, White Opaque (Part #6007290)
  • CulturPlate™-384, White, Sterile, TC-treated (Part #6007680)
• Both formats are also available in 96-well versions.

Additional materials:

• Plate lids or breathable seals (e.g., from Nunc™) to prevent evaporation
• Cells expressing the target of interest (endogenous or transfected lines). Revvity also offers frozen cell lines (e.g., AequoZen™, cAMP-ZEN™) compatible with these assays.

Assay development and optimization

To achieve optimal performance with AlphaLISA SureFire Ultra assays, several experimental parameters should be carefully optimized. Below is a summary of key variables and recommendations:

1. Cell seeding density
   • Test a range of cell densities (e.g., 10K, 25K, 50K cells/well in 96-well format).
   • Optimal density ensures sufficient signal without causing signal saturation (hook effect).
2. Recovery time after seeding
   • Allow overnight to 2 days for cells to adhere and recover before stimulation.
   • Optimal activation of some target proteins may need 2 days of culture before stimulation.
3. Serum starvation
   • Reduces basal phosphorylation levels by removing growth factors.
   • Test 0%, 1%, and 10% serum conditions for 1–3 hours or overnight.
   • Optimal conditions vary by cell line and signaling pathway.
4. Use of pathway inhibitors
   • If serum starvation is insufficient to reduce basal activity, consider adding specific inhibitors.
   • Pre-incubate for 15 minutes to 2 hours (in some instances up to 24 hours) depending on the inhibitor type and target.
5. Stimulation time course
   • Phosphorylation kinetics vary by target.
   • Suggested range: 5 minutes to 8 hours (in some instances up to 24 hours).
   • Identify the time point that yields the highest signal-to-basal ratio.
6. Agonist dose-response
   • Perform a titration to determine EC₅₀ and EC₉₀ values.
   • Use a concentration between these values for antagonist screening.
7. Incubation temperature
   • All incubation times are performed at 37°C, but room temperature can be used if needed.
   • Consistency in temperature is important for reproducibility.
8. Lysis buffer volume
   • For 96-well plates: 25–100 µL.
   • For 384-well plates: 10–50 µL.
   • Lower volumes can concentrate lysates and enhance signal.
9. Protocol format
   
        Choose between:
    
   • 1-plate protocol: All steps in one plate (ideal for high-throughput).
   • 2-plate protocol: Separate culture and detection plates (allows microscopy and multiplexing).