AlphaScreen SureFire Troubleshooting

ProblemSolution
Low signal           Ensure that the activation buffer is warmed to room temperature or 37°C and properly dissolved prior to use.
Ensure that all assay steps involving AlphaScreen® beads are performed in a low light environment, and that plates and reagents are stored in the dark during the incubations. AlphaScreen beads are light-sensitive.
Check to make sure you mixed the beads at the appropriate ratios.
Ensure that white opaque 384-well low-volume ProxiPlates™ are used for the assay if you are following the adherent cell protocol. The low volume of the reaction (11 µL) can cause problems in standard 384-well OptiPlates™.
Ensure that the incubation temperature for the assay is consistent. Temperature can affect counts.
Ensure that buffers are prepared correctly. In particular, ensure that the 5X AlphaScreen® Surefire® Lysis buffer is diluted to 1X prior to use, and that the Activation buffer is fully-dissolved prior to use.
For adherent cells, allow time for your cells to recover after plating. We recommend that you allow cells to recover for at least 16 hours before treating.
You can reduce the lysis volume and/or increase the cell number to produce more-concentrated lysates.
Check that the cell density is correct. Too high or low cell numbers can affect basal activation.
Ensure that the cell passage number is not too high, and that cells have not lost responsiveness.
Check that your samples do not contain potentially interfering material, such as biotin, antibodies, or phenol red, before lysing the cells.
Poor sensitivity      Ensure that you are following the correct order of addition. Order of addition can have a dramatic impact on assay sensitivity.
You can reduce the lysis volume and/or increase the cell number to produce more concentrated lysates.
Check that the cell density is correct. Too high or low cell numbers can affect basal activation.
If you are using a 1-step assay, consider switching to a two-step assay. See definitions on the AlphaScreen® SureFire® technology main page for more information.
Check to make sure that you mixed the beads at the appropriate ratios.
Ensure that the cell passage number is not too high, and that cells have not lost responsiveness.
Use a single-plate method for assaying the analyte. Transfer methods typically use only a portion of the total amount of plated cells.
High background    Check that the cell density is correct.
Ensure that the stimulation buffer does not contain serum, if the pathway being monitored is activated by serum.
Some pathways may have a high level of basal or constitutive activity. An upstream pathway inhibitor is often useful to determine assay window for these analytes.
Ensure that the cell passage number is not too high, and that cells have not lost responsiveness.
You can try reducing the amount of beads incorporated in the assay (see comments on Optional bead titration protocols)
Poor cell stimulation      For adherent cells, allow time for your cells to recover after plating. We recommend that you allow cells to recover for at least 16 hours before treating.
Check that the cells are confluent. When confluent, many signaling pathways — particularly those associated with growth, such as MAPK and PI3K pathways — can become quiescent and synchronized. When an agonist is introduced to quiescent and synchronized cells, they can respond uniformly.
Check cell harvesting conditions and ensure good cell viability after harvesting. Typically, cells should be maintained in log-phase growth, and harvested using Versene™ when they are 70-90% confluent.
Ensure that the pathway of interest is active in the cells, and is activated by the specific agonist used. This may vary depending on the cell line.
Try a different agonist of your receptor. Some agonists can be full agonists for some pathways, but only partial agonist for the pathway you are examining.
Ensure that the stimulant/agonist is not degraded. Prepare the stimulant/agonist fresh prior to the assay, and use a carrier protein such as BSA if necessary. Check the solubility of the agonist used, and modify the solvent used to make the stock and/or the dilution scheme if necessary.
Ensure that the cell passage number is not too high, and that cells have not lost responsiveness.
Day-to-day variation    Ensure that the incubation temperature for the assay is consistent. Temperature can affect bead counts.
Ensure that the incubation times are consistent from day-to-day. Changes in equilibrium can affect signal.
Ensure that the cell passage number is not too high, and that cells have not lost responsiveness.
Ensure that all assay steps involving AlphaScreen beads are performed in a light-subdued environment. AlphaScreen beads are light-sensitive.
Use the positive and negative control lysates to determine whether the variation observed comes from the reagents of the kit, the conditions used for detection, or the cell used in the assay.

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