Overview
The table below lists the variables that one can consider when performing an AlphaLISA™ SureFire® Ultra™ assay optimization. While most of the factors were mentioned or tested in this guide, this list contains helpful strategies and other factors to consider when optimizing a specific assay.
As a reminder, the typical criteria that should guide you through your assay optimization when testing the impact of the listed parameters will be:
- S/B, CV, Z', pharmacology (EC50 , IC50 , %efficacy, …)
- Intra-plate variation
- Inter-plate variation
- Day-to-day variation
Please be aware that absolute signal variation can vary from day to day, experiment to experiment, and reader to reader, when working with Alpha technology (due to differences in temperature when measuring the plate, pre-lighting before reading the plate, non-optimal reagent storage conditions, etc.). Data should always be analyzed primarily in terms of relative variations, compared to a control situation (e.g., nonstimulated cells).
We also recommend including measurement of the kit's control lysates in every experiment, for unforeseen troubleshooting. Running control lysates will allow Revvity to support you better and faster in such situations.
Cellular optimization
Mandatory
| Condition | Reason |
|---|---|
| Cellular model | Expression levels of the target protein, as well as basal level of phosphorylation and amplitude of response to stimuli, may vary from cell type to cell type. It may be advantageous to try a different cell type if the response is less than desired. |
| Cell seeding density | Typically, 10,000 to 70,000 cells per well are used in 96-well plates. Cell density can impact the quantity of analyte but can also modulate basal levels of phosphorylation. Keep in mind that in homogenous immunoassays, too much of the analyte can result in a reduced level of signal (hooking), such that in cases of low signal , both lower and higher cell densities need to be tested. |
| Cell stimulation time | The kinetics of protein phosphorylation can have a quick increase followed by a quick decrease, so it is important not to miss the optimal stimulation time, i.e., using a sub-optimal stimulation time can have a negative impact on S/B and assay quality. The optimal stimulation time of different phosphorylation events will often vary, even if working with the same cell type. |
| Agonist concentration | Using too low or too high concentration of agonist can reduce the S/B and overall assay quality. Agonist concentration can also impact the kinetics of the response; hence, these two factors should ideally be tested together. |
| Agonist type | Some agonists can be partial (less than 100% efficacy) or biased. It is important to select an agonist that aligns with the scope of the project pursued, to make sure the data reflects the relevant biological question. |
| Shaking to lyse cells | Once lysis buffer is added, shaking the plate to produce a homogenous cell lysate can be helpful for well-to-well reproducibility. |
| Lysis buffer selection | Some kits use different lysis buffers (A, B, or C). Each assay has been optimized to work with its specific lysis buffer provided in the kit. If running several assays in parallel from the same cell lysate, please check which lysis buffer is compatible with which kit. Lysis buffer from other suppliers can be compatible with the SureFire assay – to be checked on a case-by case basis. RIPA lysis buffer is generally not compatible with SureFire assays, unless the sample is diluted further in the SureFire lysis buffer. |
Recommended
| Condition | Reason |
|---|---|
| Recovery time before stimulation | The basal level of phosphorylation and ability to respond to stimuli can be impacted by the time cells grow in the well after seeding. Optimal time in culture for some cells/pathways can vary from four hours to up to four days. |
| Serum starvation | Basal phosphorylation can be decreased by removing growth factors or other signaling molecules from the media. Incubating cells with no (0%) or low (1-5%) serum-containing media can sometimes reduce background phosphorylation. Long-term serum starvation (> four hours) can also have a detrimental effect on some cellular pathways. It is important to select a consistent yet non-apoptotic-inducing starvation condition. This needs to be optimized on a case-by-case basis. |
| Antagonist or inhibitor treatment time | Generally, a 5-minute pre-incubation is sufficient to block cell surface receptors, and 30 minutes - 2 hours to block intracellular kinases. |
| DMSO tolerance | DMSO should normally cause no issues up to concentrations of 2%, depending on cell type and incubation times. It is advantageous to check the desired DMSO concentration in the final optimized assay. |
| Choice of agonists and antagonists | It can be helpful to use well-characterized agonist and antagonist compounds as control test compounds, to provide performance relativity. Depending on the cell type and conditions, the absolute IC50/EC50 may vary, as the pathway examined and assay conditions may lead to different maximal and half-maximal responses. |
Optional
| Condition | Reason |
|---|---|
| Temperature when stimulating cells | Cells must be stimulated in optimum conditions i.e. in incubator at 37 degrees and 5% CO2 , not on the bench. |
| Adherent vs. Suspension cell types | AlphaLISA SureFire Ultra kits work well with both cell types. Washing cells can be helpful in removing potential matrix effects, if needed. |
Hints/Reminders
| Condition | Reason |
|---|---|
| Phosphatase inhibitors | Phosphatase inhibitors are included in the SureFire Ultra Lysis Buffer (Orthovanadate, Pyrophosphate, and sodium fluoride). Additional phosphatase inhibitors may be added if necessary, but would require validation. |
| Protease inhibitors | The SureFire Ultra Lysis Buffer does not contain protease inhibitors, as they typically are not needed when working with commonly cultured cell types. However, protease inhibitors can be added for specific applications, such as working with cells very rich in proteases or working with tissue lysates. Protease inhibitors do not interfere with the AlphaLISA technology when used at standard concentrations. The following protease inhibitors are known to be tolerated by Alpha technology: Sigma Cat.No. P2714 or Roche cOmplete™ ULTRA Tablets Mini, EDTA-free, EASYpack Protease Inhibitor Cocktail Cat. No. 05 892 791 001. Other cases may exist, please inquire. |
| Medium in which cells are stimulated | Stimulation can be performed in culture medium or HBSS. Some culture media, such as RPMI, contain extremely high biotin concentrations (820 nM biotin), which can compete for the binding of the biotinylated antibody by streptavidin Donor beads used in the kit, leading to a decrease of the assay sensitivity. Therefore, cells stimulated in high biotin-containing media should be washed before cell lysis or stimulated in other culture media or HBSS. Also, some media contain unspecified growth factors or interfering agents that may affect the stimulation or assay window. |
For more details contact your local Revvity sales specialist or field application specialist.
Immunoassay optimization
Mandatory
| Condition | Reason |
|---|---|
| Activation buffer solubilization | Activation buffer components will precipitate at 4°C, and it is very important to be sure that all components are well-solubilized before use. To ensure performance, Activation buffer can be stored permanently at room temperature. |
| Activation buffer volume | Activation buffer concentration has been optimized for best assay performance. If deviations from the recommended protocol change the final percentage of Activation buffer in the immunoassay, assay performance may suffer. |
| Assay linearity | Linearity in the immunoassay largely depends on the cellular lysate concentration used in the assay, which is a factor of cells and lysis buffer quantities utilized. The ideal cellular lysate concentration should be such that the quantity of analyte to detect falls within the sensitive range of the assay. In extreme cases, using too much cellular material can result in the saturation of the detection reagents (e.g., the Hook effect, where more sample leads to less signal). |
Recommended
| Condition | Reason |
|---|---|
| Plate type | AlphaPlates may be advantageous for assay reproducibility, as they minimize well-to-well crosstalk effects and may lead to better assay reproducibility compared to white plates. ProxiPlates™ can be used when working with smaller volumes, as the sample is closer to the detector for increased signal detection. Color of the Lysis Buffer doesn't affect performance. |
| Lysis buffer* | The AlphaLISA SureFire Ultra Lysis Buffer is a mild detergent-containing buffer, formulated to avoid release of genomic DNA from the cells and clogged pipette tips. For specific cell types and protein targets, other cell lysis buffers can be investigated, which sometimes can result in a more complete release of target protein. |
| Volume of lysis buffer | To enrich protein levels, decreasing the volume of lysis buffer can be tested. A 96-well plate can be lysed in 25-100 µL lysis buffer. A 384-well plate can be lysed in 10-50 µL lysis buffer. |
| Activation buffer selection | Each assay has been optimized to work with its specific activation buffer. Activation buffers A, B, and C are not interchangeable. |
* Some kits contain assay specific Lysis Buffer B (5X)-Ultra or Lysis Buffer C (5X)-Ultra. Lysis Buffer B (5X) and Lysis Buffer C (5X) are assay specific and should not be interchanged.
Optional
| Condition | Reason |
|---|---|
| Choice of phosphoprotein target | Basal phosphorylation levels and amplitude of response will vary depending on the particular signaling pathway. For example, ERK is downstream of MEK1; therefore, higher S/B ratio is expected when detecting phospho-ERK compared to phospho-MEK. |
| Dispensing steps | Adding the Acceptor and Donor beads at the same time will reduce sensitivity and is not recommended. For best sensitivity, it is recommended to incubate the sample with the Acceptor bead mix for at least one hour before adding the Donor bead mix. |
| Immunoassay incubation time | The standard assay incubation time is one hour with Acceptor beads followed by one hour with Donor beads. This is sufficient for most applications. If greater sensitivity is desired, increasing the first incubation time (up to four hours), and the second incubation (up to overnight in the dark) can be tested. |
Hints/Reminders
| Condition | Reason |
|---|---|
| Assay consistency and reproducibility | Alpha technology is dependent upon a number of conditions, including the specific plate reader and ambient temperature. Steps should be taken to ensure consistency in using the same plate reader and maintaining room temperature (22-24 °C) for experiments. This will help improve the comparability between experiments. AlphaLISA SureFire Ultra kits are developed and manufactured such that each kit meets stringent quality criteria to ensure robust and reproducible assay windows within and between experiments, making the platform an optimal research tool from basic research through screening campaigns and translational studies as well as quality control of manufactured biotherapeutics. |
| Altering bead concentrations | The immunoassay reagent concentrations have been optimized in the standard protocol; changing the bead amounts is not recommended. Deviating from the recommended concentrations could result in reduced kit performance compared to the lot-specific kit performance provided in each kit's certificate of analysis. |
| Light sensitivity | The SA Donor beads, both the working solutions and the stock parent tube, are mildly light sensitive and should be handled with care. A typical light condition suitable for handling Donor beads is in a tissue culture or fume hood with the hood lights off, but the lights in the room can remain on. |
| Specificity | The antibodies used in the kits have been carefully selected, taking into account the target and species selectivity information from the providers of the antibodies, to generate a signal that is specific for the desired target. The specificity of the kit results from the specificity of both antibodies (i.e., a signal can be generated only when the two antibodies recognize the targets), resulting in very specific assays. However, in some situations additional evidence for the specificity of signal detection may be desired. In such situations using recombinant phosphorylated proteins, siRNAs specific to the target, or knock-out cell lines using, for example, CRISPR technology, or using peptides as competitors in the assay can be very useful to show specificity of the AlphaLISA signal. In the case of peptide competition, a phospho-peptide recapitulating the sequence containing the phosphorylated site can be used to demonstrate that the AlphaLISA signal obtained from unknown samples can be competed by such a peptide. Conversely, negative control peptides, such as a non-phosphorylated version of the target peptide, or an unrelated phospho-peptide, can be used as well. |