Alpha SureFire Assay Development
- Cell seeding density in microplates
- Incubation time after plating in assay plates
- Serum starvation requirement
- Cell stimulation time course
- Pathway inhibitor addition to reduce basal activity
- Agonist dose response
- Incubation temperature during stimulation
- Cell lysis buffer
- Lysis buffer volume
- AlphaScreen assay: 1-step vs. 2-step
- AlphaScreen assay incubation times
- Donor and Acceptor bead concentrations
The four variables of cell seeding density, recovery time after cell seeding, serum starvation, and stimulation time can be critical when setting up an assay for the first time. The Quick Guide to AlphaScreen® SureFire® Assay Optimization illustrates how to test these parameters in one straightforward experimental protocol.
Cell Seeding Density in Microplates
- Suggested range: 10,000-75,000 cells per well in 96-well format
- Starting amount: Try 3 different densities for the initial experiments
The initial screening experiments are designed to identify a cell seeding density that gives a signal window that is satisfactory to proceed with optimizing additional parameters. Once the assay has been optimized more fully, a cell titration study should be repeated to determine the optimal balance between cell culture requirements and assay performance.
Incubation Time After Plating in Assay Plates
- Suggested range: 1-2 days
- Start with: Try both 1 day and 2 days
Adherent cells need sufficient time after plating to recover and express the kinase activity of interest. This is particularly the case for cells that have been harvested using trypsin. With adherent cells, a minimum of 15 hours of incubation is necessary to achieve maximal activity of the ERK pathway.
For non-adherent cells, no recovery time is needed, but cells should be seeded for assay in a phenol red-free medium that contains low biotin, since phenol red quenches the AlphaScreen® signal and biotin can saturate the Donor beads.
Serum Starvation Requirement
- Suggested range: none to overnight
- Start with: 3 hours
Serum starvation may be necessary to reduce high basal levels of phosphorylation. Serum starvation may be beneficial or detrimental, depending on the pathway and cell line studied.
Cell Stimulation Time Course
- Suggested range: 5-60 minutes
- Start with: 5 and 20 minutes
The time course for agonist stimulation varies depending on the specific pathway and cell line being studied. For some pathways, the signal peaks within a few minutes and then declines rapidly. In other cases, the signal is maintained at a high level for up to an hour. Final optimization should include a detailed determination of the stimulation kinetic profile. The kinetic profile will also most likely be different at various agonist concentrations.
Pathway Inhibitor Addition To Reduce Basal Activity
- In some cases a high basal or constitutive activation of a pathway cannot be reduced by serum starvation. In this circumstance, an improved assay window may be achieved by the addition of a known pathway inhibitor to produce a lower signal for comparison to the stimulated response.
Agonist Dose Response
- Start with: EC100
For the initial experiments, we recommend adding the agonist at a concentration that would be expected to elicit a maximal signal. Once the cell culture and cell plating parameters have been optimized and standardized, a full dose response curve should be generated.
Incubation Temperature During Stimulation
- Suggested range: room temperature or 37°C
- Start with: room temperature
Generally, AlphaScreen® SureFire® assays can be performed by stimulating the cells at room temperature. Certain cell lines may respond better to stimulation at 37°C. The stimulation time course will vary depending on the temperature.
Cell Lysis Buffer
- Options: Standard or more aggressive lysis buffer
- Start with: Standard lysis buffer
Cell lysis for 10 minutes with gentle shaking (at 350 rpm) is usually sufficient for complete lysis. The AlphaScreen® SureFire® Lysis buffer is a very mild formulation, so that the cell shape will still be observable when viewed post-lysis under a microscope. However, all of the soluble components of the cells be released, while the genomic DNA will remain associated with the cells, which facilitates the subsequent pipetting steps. The lysis buffer contains phosphatase inhibitors; additional protease inhibitors or EDTA may be beneficial in individual cases. While not needed in most cases, a more aggressive lysis solution may be used for certain cell lines or for nuclear-associated kinases, but this will release chromatin and result in a more viscous solution that will need careful pipetting:
Lysis Buffer Volume
- Suggested range: 25-100 µL for a 96-well plate
- Start with: 100 µL
In most cases, 100 µL of Lysis buffer is satisfactory. Reducing the lysis to 50 µL may give an improved signal for analytes present in low abundance. Shaking (at 350 rpm) or other mixing during cell lysis is important to reduce assay variability.
AlphaScreen Assay: 1-step vs. 2-step
- Start with: 2-step protocol
In general, AlphaScreen assays can be formatted as 2-step bead additions or a single addition of a mixture of both beads. The 2-step protocol gives a higher signal than the 1-step protocol. The data sheet for each AlphaScreen® SureFire® assay will indicate if a 1-step assay is an option. If the assay yields satisfactory results using the 1-step bead addition, it gives the benefit of one less liquid handling step and incubation time.
AlphaScreen Assay Incubation Times
- Suggested range: 2 hours (Acceptor bead step) and 2 hrs to overnight (Donor bead step)
- Start with: 2 hours for each step
The AlphaScreen assay signal is usually sufficiently developed, so that the plate can be read after a Donor bead incubation of 2 hours. In some cases, a 4-hour or even an overnight incubation with Donor beads may give a higher signal window.
Donor and Acceptor Bead Concentrations
- Suggested range: 0.5-1X of Donor and Acceptor bead concentrations
- Start with: 1X (recommended)
If the signal-to-background ratio (S:B) or Z’ value is low, try adding a 2-fold lower concentration of the Donor beads and/or Acceptor beads. This may be helpful with some combinations of cell lines and assay targets.