by Caitlin Smith
Protein kinases play an important role in regulating almost all the basic building blocks of living systems—for example, cell proliferation, differentiation, apoptosis and the cell cycle. In addition, abnormal functioning of kinases and/or their signaling pathways commonly results in a diseased state, such as rheumatoid arthritis and chronic inflammatory diseases, cancer and diabetes. As our research technologies improve, so does the ability to fight these diseases using more biologically relevant assays. For years, we have gleaned valuable information from biochemical studies of isolated, purified kinases. Now we need to study them in cells, where they can interact with myriad surrounding factors, known and unknown, to discover how signaling networks truly work. Indeed, faster ways to screen potential therapeutic molecules targeting protein kinases (such as small molecule inhibitors) in cell-based kinase assays are evolving to meet growing demand.
It’s generally agreed that the primary factors to consider when choosing cell-based assays should include specificity, sensitivity and ease of use. Most products on the market exemplify more than one, and often all, of these qualities. And a seemingly mundane lab task—washing your sample during the assay—can have a great effect on all three qualities. So another important question to consider before choosing a cell-based kinase assay is: to wash, or not to wash? Depending on the type of sample you intend to analyze, an assay that includes washes could be either beneficial or detrimental.
To wash, or not to wash?
In some situations, washing your sample is good. Washing can reduce interfering background signals and is usually advantageous with ELISA-type assays or in-cell Western blots, for example, that use a fluorescent or horseradish peroxidase (HRP)-mediated detection signal. Another argument for wash-based assays is to wash away potentially contaminating materials when working with “dirtier” samples, such as some lysates, serum or blood.
On the other hand, there are instances in which washing is undesirable, because you can actually wash away some of the detection molecules that may be bound only lightly. Washes also may be a source of signal variation. Assays using fluorescence transfer often do not require washing because they are based on molecular proximity. Another drawback to washes is that they take more time, slowing high-throughput or drug-screening efforts.
Fluorescence resonance energy transfer (FRET)
If you are studying the interaction of a kinase with factors that might act at a distance (perhaps because of steric hindrance) or that may be confused by other proteins in the cellular environment, an assay based on fluorescent resonance energy transfer (FRET) could be a good choice. Detection of molecular proximity by the transfer of energy from one fluorophore to another provides a sensitive means to assay kinase activity. One example of this is Cisbio Bioassays’ homogeneous time resolved fluorescence (HTRF) technology, which can report the interactions of specific molecules that are within nanometers of each other. Cisbio’s cell-based kinase assays are designed to eliminate multiple-step protocols, speeding time to results.
Cisbio has developed three cell-based HTRF kinase assays for screening in kinase studies with different types of cells: Cellul’erk, HTRF Phospho Akt (Thr308) and HTRF Phospho Akt (Ser473). “Cellul’erk directly detects endogenous phosphorylated ERK1/2 to explore GPCR signaling pathways in high-throughput mode,” says François Degorce, director of marketing and communication at Cisbio Bioassays. “This efficient, functional readout targets ERK (extracellular signal-regulated kinase), whose phosphorylation can be modulated upon GPCR activation either through G-protein or β-Arrestin. Its antibody-based detection is sensitive enough for endogenous and over-expressed receptors and can be miniaturized to 1536-well formats.” Two other HTRF assays, Phospho Akt (Ser473) and HTRF Phospho Akt (Thr308), are designed to detect Akt. “These assays cover essential kinases in the different signaling pathways and are used as functional readouts of cell surface receptor activation, in particular for tyrosine kinase receptors,” says Degorce. “All three assays have already been adopted by a number of researchers for [either] kinase-based screening or as functional readouts, under assay conditions that require a high level of sensitivity—which technologies like HTRF can allow.”
In contrast to HTRF, PerkinElmer offers cell-based kinase assays that work through luminescence proximity technology. The AlphaScreen® SureFire® platform includes 75 kits developed for different targets as well as 10 kits for higher-volume assays (50 µl reaction volume). Unlike Cisbio, PerkinElmer's Alpha technology allows a greater functional distance between donor and acceptor beads, which lets researchers assay the interactions of molecules that might be far away from each other physically. “With our Alpha technology, you have a 200 nm window between donor and acceptor, which is why Alpha has been widely adopted for things like protein-protein interactions and working with large complexes like phosphoproteins,” says Eric Morreale, global product manager for Bio-discovery at PerkinElmer. “Certainly some targets, depending on the 3D conformation of where the antibodies bind, and the distances, will work fine with a TR-FRET [time resolved-FRET] platform, but others will be difficult because of the greater distance.”
Morreale says Alpha technology gives the AlphaScreen SureFire kinase assays greater sensitivity, which is especially advantageous in more biological environments where you may need to measure low levels of endogenous proteins. In addition, Alpha technology significantly amplifies the signal, which contributes to the assay’s sensitivity. “When you excite a donor bead, there is a photosensitizer that generates 60,000 singlet oxygen molecules per second,” explains Morreale. “This is transferred to an acceptor bead that then gives off an emission signal, resulting in a very high amplification of signal.”
ELISAs and IPs
Both ELISAs and IPs (immunoprecipitation assays) are mediated by specific antibodies. In kinase work, having an antibody that recognizes the phosphorylation form of your target protein can be invaluable. ELISAs and IPs can be used by kinase researchers to measure the relative expression levels of the total kinase of interest as well as its phosphorylated form. EMD Millipore offers a cell-based ELISA-type kinase assay, known as CELISA. “The CELISA format allows the researcher to detect phosphorylation events in permeabilized cells in 96-well plates,” says Sallie Cassel, director of marketing, antibodies and immunoassays at EMD Millipore. “It has the advantages of a low amount of sample required and higher throughput.” For example, Millipore’s Dual-Detect CELISA measures total and phosphorylated H2A.X in whole cells cultured in a 96-well plate format, using HRP and AP (alkaline phosphatase) for detection.
Millipore also offers an IP-based assay format for a range of different kinase targets. In these assays, the researcher immunoprecipitates the kinase of interest from a lysate and then incubates with an exogenous substrate. “EMD Millipore offers immunoprecipitation-based kinase assays in multiple readouts, including radiometric, Western blotting, colorimetric and HTRF/TR-FRET,” says Cassel. “Immunoprecipitation assays have the advantage in sensitivity and selectivity by allowing enrichment of a specific kinase of interest from large amounts of sample.”
Whole and intact cells
If you need to work with whole, intact cells, rather than permeabilized or homogenized cells, consider assays that use a different kind of reporter assay. For example, DiscoveRx offers more than 35 cell-based PathHunter® kinase assays designed for specific, and highly druggable, targets including receptor tyrosine kinases, cytosolic tyrosine kinases and downstream signaling kinases. With the PathHunter assays, a proprietary enzyme-fragment complementation technology, one can study the interaction of two proteins or detect protein translocation or protein degradation in whole cells, where normal intracellular factors and processes are intact. Furthermore, you can gather cell permeability information on your compounds.
The DiscoveRx no-wash, single-addition assays are compatible with high-throughput screening using chemiluminescent detection. Sailaja Kuchibhatla, senior vice president of business development and marketing at DiscoveRx, believes that assays should have the “flexibility to discover novel small molecule inhibitors (ATP or non-ATP pocket binders)” as well as an assay format that enables screening for functional antibodies. She stresses the importance of recognizing that artificial conditions can result in molecules that may or may not be physiologically relevant. “Currently, many small molecule inhibitor discovery programs are completed using in vitro biochemical assay platforms where purified kinases, artificial peptides and non-biologically relevant ATP concentrations are used, sometimes leading to weak or biologically irrelevant lead compounds,” says Kuchibhatla. “In some of the cases, truncated proteins with only the kinase domain are used. Finally, chemical diversity of libraries is also very limited and as such have chemicals that are predominantly ATP pocket binders, again due to limitations of the prevalent assay formats.”
Future single-cell tools?
There is much interest in the possibility of one day soon being able to study kinase activities in real time, or even within single cells, using high-content or label-free technologies. “Assays to monitor kinetics of kinase activity in live cells in real time are still in their infancy but would be valuable,” says Cassel. In addition, the ability to investigate individual cells would likely yield valuable information. “Microplate-based assays test a population of cells,” notes Morreale. “High-content technologies would be especially interesting, because you could actually hone in on a single cell within a population of cells and try to . . . detect differential activation. Those certainly would be valuable things to have in a researcher’s portfolio, in terms of an arsenal to use with kinases.”
The image at the top of the page is from PerkinElmer's AlphaScreen SureFire Phospho-ERK 1/2 assay.