Guidelines to successful optimization of AlphaLISA™ SureFire® Ultra™ assays
AlphaLISA SureFire Ultra assays offer a reliable method to quantify targeted phosphorylation events in cell-based experiments. To achieve optimal results, it’s crucial to thoroughly investigate and optimize assay conditions, especially in the initial experiments. This guide provides a structured approach to help you achieve the best response from your chosen modulator and cell line, and outlines further optimization steps for cellular and immunoassay parameters. Download the guide to ensure you get the best possible results.
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. 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. 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 new AlphaLISA SureFire Ultra Biotin-Free kits are designed to eliminate interferences caused by high biotin levels in the media.
Q. What's in the lysis buffer?
A. The AlphaLISA SureFire Ultra lysis buffer is a proprietary blend of buffers, detergents, and generic phosphatase inhibitors (orthovanadate and NaF), optimized for lysing a wide range of cells. Different kits may use specific lysis buffers (A, B, or C), each tailored to its respective assay. When running multiple assays from the same cell lysate, ensure compatibility of the lysis buffer with each kit. Lysis buffers from other suppliers might be compatible with the SureFire assay, but this should be verified individually. Generally, RIPA lysis buffer is not compatible with SureFire assays unless the sample is further diluted in the SureFire lysis buffer. Additives such as protease inhibitors or additional detergents can be included in the lysis buffer as needed for specific cells, but these should 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 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. What if I want to lyse the nuclear membrane as well?
A. The standard 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.
Q. Is the assay sensitive to DMSO?
A. DMSO should normally cause no issues up to concentrations of 2%, depending on cell type and incubation times. See typical data below.
DMSO resistance
Q. Can I quantitate the amount of phosphorylated analyte in my cells?
A. The Alpha SureFire kits are 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 “Technical Data Sheet” of each kit. A species compatibility datasheet is available, summarizing the cross-reactivity of each individual kit with different species. 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-phosphorylated ERK) as a control for a phospho-ERK measurement. Alpha SureFire Multiplex kits are available for this purpose. Another method to normalize the AlphaLISA SureFire Ultra signal response relies on the measurement of the Total Cofilin with the AlphaLISA SureFire Total Cofilin kit.
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 Revvity 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. 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.
For research use only. Not for use in diagnostic procedures.