Cookies on PerkinElmer
PerkinElmer uses cookies to ensure that we give you the best experience possible on our website. This may include cookies from third party websites. If you continue without changing your settings, we will assume that you consent to receive cookies from this website. You can change your cookie settings at any time. To learn more, please review our cookie policy, which includes information on how to manage your cookies.

Alpha SureFire No-wash Cellular Kinase Assays

 

Assay principle


Alpha SureFire® cellular kinase assays measure endogenous levels of phosphorylated cellular proteins (the "analytes") involved in various signaling pathways. PerkinElmer’s unique bead-based Alpha technology is highly versatile and can be used to study nearly any biological interaction using small molecules, large molecules, and binding partners of greatly disparate size. This unique technology has been combined with a new generation of cell-based, high throughput screening developed by TGR Biosciences, called SureFire®. The two technologies work together to provide a cell-based environment for assaying modulation of receptor activation and can also be used to measure responses of intracellular kinase inhibitors for drug discovery or fundamental research. For each Alpha SureFire kit, the phosphorylated protein is detected using sandwiching antibodies. One antibody is directed against a specific phospho-epitope on the analyte, while the other antibody is directed against another, non-phosphorylated, epitope on a distal part of the analyte.

Please note that some of the kits are detecting the "total" analyte content (i.e. detection of both the phosphorylated and non-phosphorylated forms of the analyte), to be used as a control or for normalization of the data acquired with the "phosphoprotein kits". 

alphalisa surefire phospho captsure_ASK.jpg
Alpha SureFire Assay Principle

In the assay, cells are treated with agents known or suspected to trigger or inhibit phosphorylation of the analyte and then lysed. The lysate is then mixed with the Alpha SureFire reagents:

In an AlphaScreen® SureFire® assay, Donor beads are coated with streptavidin to capture a biotinylated antibody. AlphaScreen Acceptor beads are coated with Protein A to capture the second antibody (the biotinylated antibody is carefully selected so that protein A will not capture it efficiently). In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of phosphoprotein present in the sample.

In an AlphaLISA® SureFire® Ultra assay, Acceptor beads are instead coated with a proprietary CaptSure™ agent that immobilizes the second assay antibody, labeled with a CaptSure™ tag. This eliminates potential interference from spiked antibodies and also results in better sensitivity and broader dynamic range. 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 newer AlphaLISA SureFire Ultra assay offers several advantages:

  • Simplified protocols
  • User-friendly reaction volumes
  • Lower background signal (due to the use of CaptSure technology for antibody specificity)
  • Higher signal-to-background
  • Better sensitivity
  • Compatible with the use of spike antibodies (i.e., studies using biotherapeutic antibodies, antibody blockers, etc.)
  • Best format for tissue sample analysis (due to the spectrum of the AlphaLISA Acceptor beads)
KitKit SizeRecommended plate formatFinal assay volume
AlphaScreen SureFire500 assay points384-well ProxiPlate11 µL
10,000 assay points384-well ProxiPlate11 µL
50,000 assay points384-well ProxiPlate11 µL
High volume (100 assay points)96-well 1/2 AreaPlate50 µL
AlphaLISA SureFire Ultra500 assay points384-well20 µL
10,000 assay points384-well20 µL
50,000 assay points384-well20 µL
High volume (100 assay points)96-well 1/2 AreaPlate60 µL

Top


Assay features


  • Cell-based assay, demonstrated applications on recombinant cells, primary cells, stem cells and tissues
  • Homogeneous (no wash) assay - mix components in well and read
  • Microplate-based assay
  • 96- and 384-well formats, also demonstrated use in 1536-well format
  • Well-suited for quantification of compound actions, as well as HTS screening
  • Alpha SureFire assays are based on Alpha assay technology.

Top


What do I need to run this assay?


AlphaLISA SureFire Ultra Assays

Available from PerkinElmer:

    • For non-adherent or suspension cell assays: you will need either a 384-well white OptiPlate or a sterile 384-well white CulturPlate, as desired. Both of these plates are also offered in 96-well format.
  • Plate lids (or breathable plate seal available from Nunc™)
  • Cells expressing the receptor or target of interest, either endogenously expressed, or as stably transfected cells (from PerkinElmer, or you can use your own cell line). Frozen cells (AequoZen®, cAMP-ZEN®) can also be used in this assay (see our Application Note). (Note: at the time these posters were assembled, only the AlphaScreen SureFire assays were available, but the AlphaLISA SureFire Ultra kits will work as well here).

Available from other suppliers:

  • Culture media, test compounds

Instrumentation/equipment:


AlphaScreen SureFire Assays

Available from PerkinElmer:

    • For non-adherent or suspension cell assays: you will need either a 384-well white OptiPlate or a sterile 384-well white CulturPlate, as desired. Both of these plates are also offered in 96-well format.
  • Plate lids (or breathable plate seal available from Nunc™)
  • Cells expressing the receptor or target of interest, either endogenously expressed, or as stably transfected cells (from PerkinElmer, or you can use your own cell line). Frozen cells (AequoZen®, cAMP-ZEN®) can also be used in this assay (see our Application Note). (Note: at the time these posters were assembled, only the AlphaScreen SureFire assays were available, but the AlphaLISA SureFire Ultra kits will work as well here).

Available from other suppliers:

  • Culture media, test compounds

Instrumentation/equipment:

Top


Products and catalog numbers


View our listing of relevant products and catalog numbers.

Top


Applications


Pathways

View our pathway map.

signaling_pathwaysASK.jpg
Signaling pathways

GPCR studies

Alpha SureFire can be used to study the activation or inhibition of G-protein coupled receptors (GPCRs) or other cell surface receptors by measuring downstream phosphorylation events. For example, most GPCRs linked to the activation of Gαi/o proteins will activate the ERK MAP kinase pathway, resulting in a phosphorylation of the ERK1/2 proteins, which can be detected with the corresponding Alpha SureFire kit. Most GPCRs linked to the activation of Gαi/o proteins will activate the PI3K pathway as well, leading to phosphorylation of Akt proteins. Many Gαs- and Gαq-coupled GPCRs will also lead to ERK1/2 and/or CREB phosphorylation. In such studies, the analyte that will be examined with an Alpha SureFire kit is selected according to the transduction pathways that are modulated by the receptor of interest. Note that for a given receptor, the signal transduction may be different in different cell lines, and that the selection of the Alpha SureFire kit may need to be adapted to the particular cell line used in the study. Because of this, and because different cell lines may express different levels of the analyte protein, not all cell lines will respond the same way. Therefore, cell line selection is an important consideration when designing an assay.

gpcr_signaling_ASK.jpg
GPCR signaling pathways

View our poster testing a panel of 80 GPCRs with the AlphaScreen SureFire pERK, pCREB, and pAkt kits.

 

Other receptor activation studies

Alpha SureFire kits can also be used to study activation/inactivation of other types of endogenous or recombinantly-expressed receptors by measuring downstream receptor-mediated signaling events. Examples include - but are not limited to - detection of the response of cells to insulin, PDGF, EGF, TNFalpha, stress pathway activating agents for example. Please refer to the "SureFire Validated Cells info" table for more detailed information.

    

Cellular kinase studies

Alpha SureFire can also be used to study signaling pathways and specific targets involved in signaling pathways. If you are interested in the activity of a certain kinase in a particular pathway, you can choose a downstream analyte to study. Alpha SureFire kits are named from the phosphorylated analyte being detected. As a control, you might also consider assessing the phosphorylation of an analyte upstream of your target of interest, as signaling pathways can be complex. You would typically use a couple of inhibitors, one targeting upstream of the kinase being studied, and the other targeting the kinase of interest. You would then compare how all of your tested compounds affect phosphorylation of the upstream and downstream analytes. 

Top


The first experiment


The Quick Guide to Alpha SureFire Assay Optimization has a detailed protocol that describes the first experiment we recommend you perform, complete with plate maps. There is a detailed initial protocol for adherent cells, and a detailed initial protocol for non-adherent cells. We recommend you run the relevant protocol, as written, for your first assay. You will be testing a broad range of conditions in a single assay, which will allow you to find optimal assay conditions quickly.

Top


Assay development


View more detailed information on each parameter. A number of parameters should be optimized for best results with this assay.

  1. Cell seeding density in microplates (cell number titration).
  2. Recovery time after seeding plates (suggested range: 1-2 days). For most kits, cells need sufficient time after plating to recover and get a low basal level of the pathway of interest. This is a suggested step when working with adherent cells.
  3. Serum starvation requirement. Serum starvation may be necessary to reduce high basal levels of phosphorylation. This requirement is pathway dependent and can also vary by cell line.
  4. Pathway inhibitor addition to reduce basal activity (if needed). In some cases, high basal or constitutive activity of a pathway cannot be reduced by serum starvation.
  5. Cell stimulation time course. Suggested range: 5-60 minutes. The optimal time will vary from analyte to analyte.
  6. Agonist dose-response.
  7. Incubation temperature during stimulation (room temperature is used most of the time, but 37°C can also be used if desired).
  8. Lysis buffer volume (50-100 µL for a 96-well plate).
  9. Other desired protocol variations (one-step vs. two-step assays, one-plate vs. two-plate assays, incubation times, etc.)

Top


Protocol adaptations (AlphaScreen SureFire assays)


After you have optimized your assay, you may wish to try changing the protocol that is recommended in a given manual (or you may need to decide between different protocols) 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.


ImmunoassayDonor and Acceptor beads are added in one stepDonor and Acceptor beads are added in two separate steps
Cell lysate preparationThe immunoassay is performed in the same plate that the cells are treated/lysed in1-step, one plate assay2-step, one plate assay
An aliquot of cell lysate is transferred to a new plate for the immunoassay1-step transfer assay2-step transfer assay

Top

 

Quick reference protocols (AlphaScreen SureFire assays)


Top

 

Volumes for making bead mixes (AlphaScreen SureFire assays)


View detailed information on volumes to use when preparing buffer/bead master mixes when running between 50-500 wells. The tables list recommended volumes of each component, based on the number of assay points (wells) you will be using.

Top


Guides and scientific posters


  • Quick Guide to Alpha SureFire Assay Optimization - illustrates how to test the four variables of cell seeding density, recovery time after cell seeding, serum starvation, and stimulation time in one experimental protocol. These parameters can be critical when setting up an assay for the first time.
  • AlphaScreen SureFire User Guide — describes kit contents, protocol options, and assay optimization
  • Cell lines validated for Alpha SureFire assays
  • Poster for AlphaLISA SureFire Ultra and SureFire Multiplex assays
  • Application note - A Practical Approach to Cell Signaling Pathway Analysis, from sensitive western blots to quantitative assays of multi-target pathways using AlphaScreen SureFire assays
  • Application Note describing SureFire multiplexing assays for total and phospho protein target
  • Application Note using SureFire assays as functional assays for downstream targets of extracellular matrix protein modulation by TGFb
  • Poster describing receptor activation assays with multiple kits
  • Poster describing assay development for TNFα receptor studies using the p38 MAPK kit
  • Poster describing CCR7 and GAL1 receptor studies using PerkinElmer gamma-irradiated FroZen™ cell lines
  • Poster describing a rapid multi-parameter optimization method for AKT phosphorylation assays, employing the JANUS® Automated Workstation

Top


Citations


View a partial listing of Alpha SureFire citations. To search our database of citations referencing PerkinElmer products, please visit the PerkinElmer Life Sciences Citations Library.

Top


Tips and FAQs


Click here for Alpha SureFire tips and FAQs.

Top


Troubleshooting


View our troubleshooting tables.

Top


Other PerkinElmer kinase technologies


Top