Overview

Basic step-by-step assay protocols are provided in the manual for each assay. An overview of the different protocols can also be found in the AlphaScreen® SureFire® User Guide. The first time you run the assay, you should follow the protocol recommended in the kit manual. Each manual has a recommended protocol, specific to the particular kit. Please note that there is a protocol for adherent cells, as well as a protocol for non-adherent or suspension cells.

After you finish optimizing your assay, you may wish to try changing the protocol, depending on what your needs are. For example, if you are automating your assay, you may wish to perform your adherent cell assay as a single-plate assay. This page describes the differences between one-step and two-step addition protocols, the difference between one-plate and two-plate (transfer) protocols, the difference between adherent vs. suspension cell protocols.

Cell treatment for AlphaScreen SureFire

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Types of assay protocols

• 1-step assay: An assay format where both the Donor and Acceptor beads are added at the same time to your cell lysate. This protocol is convenient due to the reduction in time and number of steps of the assays; however, it is recommended only for a few kits (pERK, pMEK) due to the variable reduction of signal that is observed in the 1-step assay, compared to the 2-step assay.
• 2-step assay: An assay format where you first add Acceptor beads and antibodies to your cell lysate and incubate for 2 hours, then add Donor beads and incubate until the plate is read. The 2-step assay is always more sensitive than the 1-step assay. How dramatic this difference is will depend on the kit you are using.

1-step vs. 2-step assay protocols

• 1-plate assay: An assay where cells are treated and lysed in the same plate to which antibodies, Donor beads, and Acceptor beads will be added. You will not be transferring your cell lysate to a separate plate. This may be preferable when automating the assay for high throughput applications. All non-adherent/suspension cell protocols are written as 1-plate assays. In the one-plate assay, a single analyte can be analyzed in the cell lysates.
• 2-plate assay: An assay where cells are treated and lysed in one plate, then an aliquot of the lysate is transferred to a second plate for addition of the beads. This can be useful if you will be testing the same lysate against several AlphaScreen® SureFire® analytes, and is the recommended protocol for assay development.

One-plate protocol overview

• Adherent cell protocol: A protocol that is recommended if you are working with adherent cells.
• Non-adherent cell protocol: A protocol that is used if you are working with suspension/non-adherent cells. Under this assay format, particular care must be taken to the medium used (for example, avoid medium containing serum and too much biotin due to their potential interference with the AlphaScreen® SureFire® antibodies binding).

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Optional bead titration protocols for improving the signal-to-background ratio (S:B)

If you would like to try to improve the S:B ratio of an AlphaScreen® SureFire® kit, you may perform a bead titration. This involves performing the assay using the beads at the standard concentrations, then comparing the results using the beads at a 1:2 or a 1:4 dilution. This can sometimes reduce the background more than the signal, thereby improving the S:B ratio and the Z’ value of the assay.

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