Alpha SureFire Tips and FAQs
Q. Where are the antibodies?
A. The antibodies are in the reaction buffer. This is why we recommend that you be very careful when storing the reagents in this kit. When the reaction buffer is mixed with the Acceptor beads, one of the antibodies will associate with the Acceptor beads, and when the Donor Beads are added later, they will capture the biotinylated antibody.
Q. For AlphaScreen® SureFire®, wouldn't both antibodies associate with the Protein A-coated bead, ruining my assay?
A. No, the antibodies are selected so that only one of the antibodies will efficiently bind to the Protein A-coated bead.
Q. How does CaptSure™ tag technology work?
A. AlphaLISA® SureFire® Ultra™ assays use CaptSure technology to capture antibody to the Acceptor beads. The CaptSure tag is a peptide, covalently linked to the antibody that recognizes the target-of-interest. The CaptSure binding protein (on the AlphaLISA Acceptor bead) is a monoclonal antibody (covalently conjugated to the bead). This bead-conjugated antibody specifically recognizes the CaptSure peptide tag on the target-specific antibody.
Q. If I am currently using an AlphaScreen SureFire kit, how do I adapt my assay to the equivalent AlphaLISA SureFire Ultra kit?
A. As the dynamic range of the AlphaLISA SureFire Ultra kits is very wide, you do not necessarily need to change the number of cells used per well. However to reach the optimal S/B we recommend titrating your cells (or cell lysates). Optimal performance may be achieved by the use of a lower amount of cell lysates (fewer cells) in the AlphaLISA SureFire Ultra kit compared to the AlphaScreen Surefire assay. You may keep the agonist concentration and time of stimulation unchanged.
Q. Are the AlphaLISA SureFire Ultra and AlphaScreen SureFire Donor and Acceptor beads the same?
A. Both technologies use the same type of streptavidin-conjugated Donor bead (provided at 2 mg/mL stock concentration for the AlphaLISA SureFire Ultra format, and at 5 mg/mL for the AlphaScreen SureFire format). However, the Acceptor beads are different: the AlphaScreen SureFire assay uses Protein A-conjugated Acceptor beads. The AlphaLISA SureFire Ultra assay uses CaptSure-binding protein-conjugated Acceptor beads.
Q. Is it helpful to try using different beads concentrations?
A. The bead concentrations and antibody concentrations have already been optimized in the AlphaLISA SureFire Ultra kits, and we do not recommend changing these concentrations. It is expected that increasing beads concentrations will lead to increased levels of background signals. The only case where the concentrations of Streptavidin Donor Beads may benefit from an increase is when working with samples containing a high biotin concentration, like RPMI-1640 medium, or some tissue lysates. In that case, you can use the 2 mg/mL SureFire Ultra AlphaScreen Streptavidin Donor Beads (the 5 mg/mL Streptavidin Donor beads will work as well).
Q. Can I measure AlphaLISA SureFire Ultra and AlphaScreen SureFire assays on the same instrument?
A. All PerkinElmer plate readers (EnVision, EnSpire, EnSight) can utilize the same instrument program to measure both technologies. If you are using a plate reader from a different vendor, please inquire with the instrument manufacturer whether the instrument can measure both AlphaScreen and AlphaLISA signals.
Q. What's in the lysis buffer?
A. Both the AlphaScreen SureFire and AlphaLISA SureFire Ultra lysis buffer are a proprietary mixture of buffers, detergents, and generic phosphatase inhibitors (orthovanadate and NaF), optimized for lysis of a broad range of cells without releasing nuclear DNA. Additives can be supplemented to the Lysis buffer as required for particular cells, and may include excipients such as protease inhibitors or extra detergents. These will need to be tested on a case-by-case basis.
Q. Are there protease inhibitors in the lysis buffer?
A. No, there are no protease inhibitors in the AlphaScreen SureFire or AlphaLISA SureFire Ultra lysis buffers. Additives can be supplemented to the Lysis buffer as required for particular cells, and may include excipients such as protease inhibitors or extra detergents. These will need to be tested on a case-by-case basis.
Q. Are the AlphaLISA SureFire Ultra and AlphaScreen SureFire buffers the same?
A. No, the lysis buffer, activation buffer, and reaction buffers differ. The lysis buffer in the AlphaScreen SureFire kit is compatible for use with Protein A-coated Acceptor beads. Alpha SureFire Ultra lysis buffer should not be used in AlphaScreen SureFire assays. The reaction buffer contains the antibodies required for each assay. The AlphaLISA SureFire Ultra reaction buffer contains biotinylated antibody and antibody tagged with CaptSure. The AlphaScreen SureFire reaction buffer does not contain a CaptSure-tagged antibody. Therefore,the reaction buffers are not interchangeable.
Q. What if I want to lyse the nuclear membrane as well?
A. In most cases, the standard SureFire Lysis buffer will work. However 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. See the “Cell Lysis” section below to learn how to run this more aggressive Lysis protocol.
Q. Is the assay sensitive to DMSO?
A. In general, we observe good DMSO-resistance for the SureFire Ultra kits. See typical data below.
Q. Can I quantitate the amount of phosphorylated analyte in my cells?
A. The Alpha SureFire kits were designed for detecting increases or decreases in phosphorylation upon cell treatment, and not for absolute quantification of the phosphorylated protein content. You may be able to find recombinant phosphorylated analyte from other companies to set up a standard curve. We do not carry such standards at this time.
Q. What types of cells can be used in the assay?
A. The assay can be used for many adherent and suspension cell types, including transfected cell lines, primary, and stem cells. If you can detect your phosphorylated analyte in your human cell line with a western blot, Alpha SureFire should work for you.
Q. Will the kits detect phosphorylated proteins from other species?
A. Most of the kit antibodies should detect the phosphorylated analyte from rat and/or mouse. This information is provided in the “kit-Specificity Information” section of the manual for each kit. If you need more information about species specificity, please contact technical support.
Q. What concentration of lysate is required?
A. Typically, the assay works well in the range of 0.2-0.5 mg/mL of lysate.
Q. Can cell lines with stable or transiently-transfected kinases or receptors be used?
A. Yes, both transient and stable cell lines have been shown to elicit good responses; however, we recommend using stable cell lines rather than transiently-transfected cell lines to enhance assay reproducibility.
Q. How should the cells be handled?
A. Cells should be harvested from flasks for seeding into microplates when they are approximately 70-90% confluent. Cells should be detached from the flasks using mild conditions (Versene™ or mild trypsinization), accurately counted, and diluted to the appropriate density in fresh media. If using adherent cells, plate for at least 15 hours prior to assaying, to allow cells to regain full signaling capacity after harvesting.
Q. Do I have to worry about sterility when plating the cells and allowing them to recover?
A. When we perform adherent-cell assays, we plate overnight (~15 hours) with a medium. Since bacterial growth can be quite rapid, we advise using the usual cell culture precautions to keep all the material and medium sterile at this step. So you should use sterile TC-treated microplates, such as our TC-treated ProxiPlate or CulturPlates.
Q. How can I normalize the assay, with respect to number of cells?
A. The GAPDH Alpha SureFire assay has been designed to measure endogenous cellular GAPDH in cell lysates, as a reference housekeeping gene, and can be used in conjunction with the other Alpha SureFire assay kits for the screening of both modulators of receptor activation (e.g. agonists and antagonists) as well as agents acting intracellularly, such as small molecule inhibitors of upstream events. The GAPDH kit allows for normalization of cell number in different samples when using other Alpha SureFire assay kits to measure phosphorylated targets. Another possibility is to use one of the “total” assay kits, for example for measuring total ERK protein (i.e. phosphorylated + non-phopshorylated ERK) as a control for a phospho-ERK measurement. Alpha SureFire Multiplex kits are available for this purpose.
Q. Are Alpha SureFire assays scalable?
A. There are validated protocols that are scalable down to a 4-5 µL total reaction volume in 1536-well plates. This will need to be tested on a case-by-case basis. If you are using adherent cells, do not have shallow-well plates, and need to scale up your reaction, simply double the volume of the cell lysate (or control) and bead mixes being added to your well. Your reaction volume will change proportionally. In any case, we do not recommend changing the relative proportions of the assay components, as over-diluting the reagents may result in altered assay characteristics.
Q. Can I assay multiple analytes from a single lysate?
A. Yes, this is a unique feature of Alpha SureFire protocols. Because you will lyse in 50-100 µL of lysis buffer, you can test multiple analytes with aliquots from the same lysate.
Q. Can I incubate my assays overnight?
A. The assay time recommended for each kit enables you to get a signal close to the optimum after the specified incubation period. However, incubating for longer periods, up to overnight at room temperature, may in some cases increase the assay window further.
Q. What do I do if PerkinElmer does not have my analyte?
A. In order to benefit from our experience and from our proprietary reagents developed to optimize the detection of phosphorylated proteins, you can ask us to develop this assay for you: contact your local representative or email our technical support teams. You can also develop your own Alpha assay. Please refer to the Create your own Alpha assay section. Finally, you may find in the signal transduction pathway another target, located upstream or downstream your initial choice, and already available as a catalog item, that may fit your needs as well.
Q. I will be treating my cells with an antibody, and I am worried that this will bind to the Protein A-coated Acceptor beads and interfere with the assay. Is there another bead option?
A. We offer AlphaLISA SureFire Ultra kits, which use CaptSure-coated Acceptor beads (instead of Protein A Acceptor beads). These AlphaLISA SureFire Ultra kits are specifically designed to be resistant to test antibodies. Another option, not as perfect but immediately available, may be to replace Protein A beads by anti-species specific IgG Acceptor beads. Contact your local representative or email our technical support teams to get our help on achieving this.
Tips for users new to Alpha technology
- AlphaScreen and AlphaLISA Acceptor beads are somewhat light-sensitive. We recommend that you work with the beads under dim lighting conditions, and keep plates covered with our black seal (Cat. No. 6050173) or foil during incubation and preparation steps.
- The Alpha signal is temperature-dependent. You will want to keep the temperature of your incubations and the temperature during your plate reads consistent from day to day to avoid raw count variation.
- Culture media containing high concentrations of phenol red or FBS (or other sera) can interfere with an AlphaScreen assay. If you are following our suspension cell protocol, you may need to use a medium that does not contain phenol red. You also may need to keep your FBS concentration at or below 10%. When following the adherent cell protocol, the instructions direct you to aspirate the culture medium prior to cell lysis, so you do not need to worry about interference from your culture medium. An alternative is to use the AlphaLISA SureFire Ultra kits.
- Culture media containing high concentrations of biotin (such as RPMI) can interfere with Alpha assays using Streptavidin. If you are following our suspension cell protocol, you may need to use a medium that does not contain high biotin concentrations, or HBSS buffer. When following the adherent cell protocol, the instructions direct you to aspirate the culture medium prior to cell lysis, and maybe even wash the cells before their lysis, so you do not need to worry about interference from your culture medium. An alternative, within certain limits, is to increase the concentration of Streptavidin Donor beads in the assay.
- Keep in mind that Protein A beads can bind many classes of antibody. If you are using antibodies in your studies, you should use an AlphaLISA SureFire Ultra kit instead of an AlphaScreen SureFire kit to avoid the use of Protein A beads.
General cell handling
Cells should be harvested from flasks for seeding into microplates when approximately 70-90% confluent. The cells should be detached from the flasks using mild conditions, accurately counted, and diluted to the appropriate density in fresh media. If using adherent cells, allow to adhere in full media for at least 6 hours prior, allowing time for cells to regain full signaling capacity after harvesting.
Serum starvation requirement
Some applications may benefit from a serum-starvation step, where full media is removed and replaced with serum-free media. This can reduce the basal level of activity of certain signaling pathways, such as MAPK signaling. This step should be optimized on a case-by-case basis, but will generally be from 2 hours, up to overnight. Be careful as some applications may perform better in the absence of serum starvation, and our recommendation is that both assay conditions are compared when setting up an assay.
The standard Lysis buffer is of a gentle nature, and cells will often appear "intact" when viewed with a microscope. However, the soluble components of the cells have been released. A more aggressive lysis formulation can be prepared by the addition of activation buffer to the lysis buffer formulation, which will solubilize the cells more thoroughly and release more proteins bound in protein complexes. The more aggressive lysis buffer can easily be prepared prior to lysis by engaging the activation buffer at the cell lysis stage. To achieve this, you can:
- For AlphaScreen SureFire assays: dilute Activation buffer 5‐fold in 1X Lysis buffer (e.g. dilute 1 mL SureFire Activation buffer in 4 mL 1X Lysis buffer), and then mix 5 µL of cell lysate with Activation Buffer + 4 µL of Acceptor Mix (2 hours) + 2 µL of Donor Mix (2 hours) (which is different from the standard protocol, 4 µL / 5 µL / 2 µL).
- For AlphaLISA SureFire Ultra assays: dilute Activation buffer 50-fold in 1X Lysis buffer (e.g. dilute 100 µL SureFire Ultra Activation buffer in 4.9 mL 1X Lysis buffer), and then mix 10 µL of cell lysate with Activation Buffer + 5 µL of Acceptor Mix (1 h) + 5 µL of Donor Mix (1 h) (i.e., the same volumes as in the standard protocol).
The release of chromatin may be observed using this lysis step, which may make the lysates more difficult to handle.
! Important: if Lysis buffer/Activation buffer mix is used to lyse the cells, ensure that no Activation buffer is added to the Acceptor mix during preparation (i.e. Acceptor mix should contain just Reaction buffer and AlphaLISA beads).
A low signal can often be improved by generating more concentrated lysates. In most cases, a typical adherent cell line grown in 96-well plates is readily detected in a lysis volume of 50-100 µL. However, for low abundance proteins, the lysis volume can be adjusted down to 25 µL, to increase the analyte concentration in the lysate. Cells that express very low levels of the target of interest (e.g. if immunoprecipitation is required to see a band on a Western blot) then it may be below the detectable limit for SureFire assays.
The standard Lysis buffer supplied with the kits contains phosphatase inhibitors. The addition of protease inhibitors or EDTA may be beneficial in some cases.
Assay incubation times
The general assay incubation times that are recommended are 1 hour after each assay reagent addition in the AlphaLISA SureFire Ultra assay format (with the exception of kits that can be run using a “single incubation” protocol, where a single 2 hour incubation is used after adding the two reagents) and 2 hours after each assay reagent addition in the AlphaScreen SureFire assay format. Longer incubations (up to overnight) may be more convenient for certain assays, and can enhance sensitivity in some cases.
Alpha beads concentrations
The standard concentration of Alpha Donor and Acceptor beads is provided. However, if poor sensitivity is observed, adjusting the bead concentrations in the Donor Mix may help. For the AlphaScreen SureFire format, adjusting the bead concentrations in the Acceptor Mix may be explored as well.
The AlphaScreen SureFire assays are compatible with most cell culture media and reagents, however there are some exceptions. Media that contain biotin (i.e. RPMI) will reduce assay sensitivity due to the interference of biotin on the antibody-streptavidin interaction. When it is necessary to use a media such as RPMI for growing cells, they should be harvested and resuspended in HBSS or similar buffers for the assay, or adherent cells should be washed before lysis.
|Biotin (present in media such as RPMI at concentrations as high as 820 nM, while most culture media contain 15 to 30 nM biotin)||Can compete for streptavidin binding of the biotinylated antibody||Use another culture media|
|Wash cells before lysis|
|Increase Streptavidin Bead concentration|
|Dilute cell lysate before testing|
|Serum||Can interfere with immunoassay components||Use serum-free culture media – and/or|
|Wash cells before lysis – and/or|
|Dilute cell lysate before testing|
|SDS||Can denature streptavidin already at low concentrations||Change cell Lysis method to avoid or decrease SDS content|
|Dilute cell lysate before testing|
Phenol red and antibodies are not expected to interfere with the AlphaLISA SureFire Ultra assay format.
Cell types that can be used in the assay
The assay can be used for many adherent and non-adherent cell types, including transfected cell lines, primary, and stem cells. However, because phosphorylated protein expression and phosphorylation conditions can vary from one cell line to another, some cells may be more amenable for particular assays than others. Parameters such as stimulation time and cell number should be optimized for each cell line used.
Cells over-expressing a receptor of interest have been shown to elicit good phosphorylation responses. Cell lines expressing high levels of an intracellular kinase of interest can also be used, but should be full-length to ensure correct binding of assay antibodies. When using overexpressed intracellular targets, the concentration of cell lysate should be optimized to ensure the signal is within the working range of the assay.
The primary AlphaScreen SureFire and AlphaLISA SureFire Ultra assay methodologies have been optimized respectively for a low‐volume 384‐well Proxiplate and Optiplate-384 plates. However, the assays are scalable down to 4‐5 μL total assay volume in 1536‐well microplates, allowing a saving of both lysate and assay reagents.
|Assay||Item||Catalog number||Number of plates|
|AlphaScreen SureFire 11 µL||Proxiplate™‐384 Plus, white, shallow well assay plate||6008280||50|
|AlphaLISA SureFire Ultra 20 µL||Optiplate™-384, White Opaque assay plate (1)||6007290||50|
|AlphaPlate-384, Light gray assay plate (2)||6005350||50|
|Alpha SureFire HV kits||White 96-well 1/2 AreaPlate||6005560||50|
|Cell culture and 11 or 20 µL assay||CulturPlate-384, white, sterile, with lid, 384-well (3)||6007680||50|
|Cell culture, with transfer of cell lysate to another plate for immunoassay||ViewPlate-96 F, TC, black frame with clear bottom, sterile, with lid, 96-well (4)||6005182||50|
|4 – 5 µL assay||AlphaPlate 1536-well||6004350||50|
More information on plates at www.perkinelmer.com/microplates.
Choosing an assay protocol
Transfer assay methods are those where the cells are grown in microplates, typically either 96-well or 384-well, stimulated/inhibited and lysed. An aliquot of this lysate is then transferred to an assay plate to analyze for a particular phosphoprotein. This format is particularly useful for method development and optimization, low to medium throughput projects, and when assaying for multiple phospho-proteins from a single well. Single plate methods are usually for high-throughput projects, where wells are analyzed for a single target, and minimal use of reagents and liquid handling equipment is essential.
Assaying for multiple targets from a single lysate
One of the unique features of SureFire protocols is the use of very small amounts of cell lysate. The standard protocol suggests the use of just 4 or 10 µL of lysate per well, whereas a typical 384-well or 96-well cell culture microplate would use 20-100 µL of lysis buffer per well. Therefore, a typical cell lysate can be assayed for many targets, taking into account that temporal and expression level constraints can vary from cell line to cell line. The wider dynamic range of the AlphaLISA SureFire Ultra assays (compared to AlphaScreen SureFire) assays can be an advantage when wanting to analyze phospho-proteins that have very different levels of expression in the cells analyzed.
Subtracting a background control for data analysis
In most cases, we would not recommend the subtraction of buffer-only background during data analysis. For methods such as ELISA, subtraction of buffers-only controls is possible because cellular debris and interfering substances are washed away during the many wash steps involved in typical ELISA protocols. In contrast, SureFire assays are homogeneous, and the assays are performed and read in crude cellular lysates containing proteins, lipids, nucleic acids and other cellular debris. Therefore, in this homogeneous system, the most appropriate background control for subtraction is a cellular lysate that has no phosphorylated target. Subtraction of cellular background/basal phosphorylation prior to analysis may be useful in some instances.