Novel detection method for chemiluminescence derived from the Kinase-Glo luminescent kinase assay platform: Advantages over traditional microplate luminometers.

The efficacy of cellular signal transduction is of paramount importance for the proper functioning of a cell and an organism as a whole. Protein kinases are responsible for much of this transmission and thus have been the focal point of extensive research. While there are numerous commercially available protein kinase assays, the Kinase-Glo luminescent kinase assay (Promega) provides an easy-to-use and high throughput platform for determining protein kinase activity. This assay is said to require the use of a microplate spectrophotometer capable of detecting a luminescent signal. This study shows that:•The ChemiGenius Bioimaging system (Syngene), typically used for visualizing chemiluminescence from Western blots, provides an alternative detection system for Kinase-Glo luminescence.•The novel detection system confers an advantage over traditional luminometers, in that it allows visualization of the luminescent wells, which allows for the real-time analysis and correction of experimental errors (i.e. bubble formation).•Determining kinase kinetics using this detection system produced comparable results to previous studies on the same enzyme (i.e. glycogen synthase kinase 3).

Materials and methods

Kinase-Glo standard curve

ATP standard curves were created using both the Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, ON, Canada) and the ChemiGenius Bioimaging System (Syngene, MD, USA). Using either machine, Mg-ATP (made in Tris–HCl, pH 7.5) concentrations were varied between 0 and 100 μM (as suggested by the Kinase-Glo Plus platform protocol), and made up to a final volume of 50 μL in a 96-well Costar opaque black microplate or to a final volume of 20 μL in a 384-well Costar opaque black microplate. Equal volumes of Kinase-Glo Plus reagent was added to the wells containing ATP and these solutions were allowed to sit for 10 min to allow for the luminescent signal to stabilize. Detection using the Cary Eclipse spectrophotometer involved selecting single read mode for a luminescent signal at 550 nm (experimentally determined luminescent λmax) and detecting that signal for 1 s. In contrast, utilizing the ChemiGenius Bioimaging system, no specific filter was chosen and luminescence was measured for 1 s. Accompanying GeneTools software was used to measure the luminescence of each well.

Method validation using glycogen synthase kinase assays

Kinetic assays using a skeletal muscle-derived protein kinase, GSK3, were conducted using the ChemiGenius Bioimaging System. To do so, GSK3 was partially purified from the skeletal muscle of 13-lined ground squirrels using ion exchange and affinity chromatography (specifics of purification procedure given in Appendix A).

Those fractions that contained GSK3 were subsequently pooled and spun in centrifugal filters (10 kDa cut-off; Amicon) at 8000 RPM for 20 min at 5 °C. The resulting concentrate was then used in GSK3 assays, which contained 50 μM ATP, 5 mM MgCl2, GSK3 peptide (YRRAAVPPSPSLSRHSSPHQ(pS) EDEEE; SignalChem) ranging from 0 to 185 μM, and 25 mM Tris–HCl, pH 7.5. The peptide is phosphorylated at the indicated serine residue as GSK3 will preferentially phosphorylate substrates that are previously phosphorylated by other kinases. The underlined serine residues in the above peptide can both be phosphorylated by GSK3 [1]. Selective addition of 1 mM LiCl was used to inhibit GSK3 activity and act as a negative control. Assays were attempted in both 96-well and 384-well Costar black microplates, and kinase reaction was allowed to proceed for 1 h at room temperature prior to the addition of the Kinase-Glo reagent. Again, once the Kinase-Glo reagent was added, the reaction wells were allowed to sit for 10 min so that the luminescent signal stabilizes. Luminescence was detected as mentioned above for the ATP standard curve.

GSK3 activity and Km calculation

Luminescent units determined using the GeneTools software were converted to GSK3 activities by first determining the concentration of ATP that remained after the kinase reaction. This is subsequently converted to the concentration of ATP utilized in the kinase reaction. This value was then divided by the assay time and the amount of protein present in each assay. Protein content of enzyme preparations was determined with the Bradford assay using the Bio-Rad prepared reagent and bovine serum albumin as the standard. These activities were then used to determine the Km peptide with the help of the Kinetics v.3.5.1 computer program [2].

Additional information

The Kinase-Glo luminescent kinase assay (Promega) is a high-throughput method for detecting protein kinase activity by quantifying the amount of ATP remaining following a kinase reaction. The luminescent signal is produced via the reaction shown below:Typically these assays are conducted in opaque microplates with luminescence being detected using a microplate spectrophotometer capable of detecting a luminescent signal (for recent examples see [3,4]). If not already present within your laboratory, purchase of such a spectrophotometer would be significantly costly. For this reason, alternative detection systems may be extremely useful in broadening the use of this assay platform, especially if this equipment is common to many labs. The ChemiGenius Bioimaging system (Syngene) is typically used in detecting fluorescent or chemiluminescent signals from Western blots or electrophoretically separated PCR products (for recent examples see [5,6]). This study adapts the ChemiGenius Bioimaging system for the Kinase-Glo platform, validates its use for determining protein kinase activity, and indicates the advantages of using this Bioimaging system over other luminometers.

Utilization of the ChemiGenius Bioimaging system for the purpose of detecting the luminescent signal produced by the Kinase-Glo luminescent kinase assay requires that the assay will function as it would in a traditional luminometer. The first step in assessing assay suitability is to determine the linear dynamic range for the assay using the ChemiGenius Bioimaging system as compared to that observed with a luminometer. The Kinase-Glo Plus assay platform claims to have a linear dynamic range from 0 to 100 μM ATP and this is reflected in standard curves created using the Cary Eclipse fluorescence spectrophotometer and the ChemiGenius Bioimaging system (Fig. 1). Both standard curves elicited R2 values above 0.99. Given the linearity of the Kinase-Glo Plus assay platform, kinetic analyses were undertaken for GSK3 to determine the suitability of the ChemiGenius Bioimaging system for kinase assays.

Fig. 1

ATP standard curves for Kinase-Glo Plus luminescent kinase assay platform. (A) The ATP standard curve when utilizing the Cary Eclipse fluorescent spectrophotometer, and (B) the ATP standard curve when utilizing the ChemiGenius Bioimaging system. All data were collected using 96-well black microplates. Data are mean ± SEM, n = 3.

GSK3 was partially isolated from the skeletal muscle of 13-lined ground squirrels using a combination of ion exchange and affinity chromatography. GSK3 Km peptide was subsequently determined to be 39 ± 4 μM when using the ChemiGenius Bioimaging system for luminescence detection (Fig. 2). The Km obtained in this study is similar to those determined in studies that utilized other detection techniques. For instance, a study of wood frog GSK3 found the Km peptide to be 23 ± 4 μM when utilizing radioactive ATP and subsequent detection of radiolabelled peptides on a phosphor screen [7]. In addition to the seemingly accurate Km curve generated, the ChemiGenius Bioimaging system provided a unique ability to visualize the end point luminescence after the kinase reaction (Fig. 2B). This advantage over traditional luminometers allows for well-by-well assessment of experimental error, and for the potential to correct those errors in real-time. For instance, eliminating small bubbles, that would otherwise be unseen in luminometers, increases the accuracy and precision of luminescent measurements. It should also be noted that this type of chemiluminescent detection is not limited to the ChemiGenius Bioimaging system, but likely many bioimagers that were designed to detect chemiluminescence from Western blots or PCR gels. Individual machine settings will vary, but the novel detection of kinase activity via the Kinase-Glo platform should be widely applicable.

Fig. 2

Km peptide for GSK3 derived from the skeletal muscle of 13-lined ground squirrels. (A) The kinetic curve for the determination of Km GSK3 peptide and (B) A representative sequence of luminescent wells that were used to generate kinetic curves shown in (A). All data were collected using 96-well black microplates. The data representative in graph are mean ± SEM, n = 3 on independent determinations on separate enzyme samples.