What are the common methods available to detect kinase activities?
Posted May 24, 2023
Common methods available to detect kinase activities include:
Radiometric Kinase Activity Assay
A traditional technique for detecting kinase activity, radiometric protocols involve the use of a 32P-labeled ATP. The steps can be adapted for use with immunoprecipitated cultured cells or with recombinant affinity-tagged purification over different reaction conditions.
To assess kinase activity, a buffer made using ATP, a protein inhibitor, and reducing agent, is combined with the kinase sample and incubated. A stop solution is added and SDS-PAGE is then performed. Upon analysis, kinase activity is calculated in units of nanomole phosphate transferred per min per mg kinase.
Qualitative Polymerase Chain Reaction (qPCR)
A highly sensitive and versatile technique, Qualitative polymerase chain reaction (qPCR) is capable of quantitating multiple kinases simultaneously on a single panel with high throughput capabilities. This allows researchers to detect the associated DNA label and compare it to known controls to determine the amount of kinase captured in test samples. qPCR can also be used to determine dissociation constants for kinase interactions and to detect type I, type II, and allosteric kinase inhibitors.
A significant advantage of using qPCR to detect kinase activities is that it does not require ATP, which allows researchers to measure only true thermodynamic interaction affinities rather than relying on IC50 values like most other techniques.
There are multiple fluorescent methods that can be used to detect kinase activity without the use of radioactivity.
- Most commercially available kits utilize simple fluorescence endpoint measurements, where fluorescent probes bind and label peptide substrates. These assays can be studied in real time and offer a powerful tool for screening compound libraries for kinase modulators, which can be beneficial for finding new treatments
- Using Fluorescent polarization (FP), another method to detect kinase activity, researchers can analytically graph kinase activity as a function of concentration versus polarization, where the amount of phosphorylated substrate present is inversely proportional to the amount of antibody bound to the tracer.
- Time-resolved (TR) fluorescence resonance energy transfer (FRET) is another method of detecting kinase activity. In this technique, resonance energy transfer occurs when the donor and acceptor fluorophores are brought into close proximity to each other. FRET analysis can be carried out by measuring changes in the intensity of the acceptor probe's sensitized demission or by monitoring the decrease in the lifetime fluorescence of the donor probe.
- Another fluorescent detection method, Fluorescence lifetime microscopy (FLIM) is a versatile imaging technique that utilizes differences in the exponential rate of decay of photon emission from a fluorophore in a sample. FLIM works by measuring the average fluorescence lifetime (the average time that fluorescent molecules spend in an excited state). FLIM can also indirectly measure molecular concentrations and interactions related to the fluorescence lifetime of the fluorophores.
Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
Liquid Chromatography-Mass Spectrometry are phosphoproteomic techniques that enable the unbiased identification of phosphorylation by highly selective enrichment of phosphopeptides. The amount of phosphorylated proteins and phosphosites in a sample determines the observability of kinase activity. LC-MS/MS enables large-scale identification of phosphorylated peptides and phosphosite localization, providing crucial information on the relationship between kinase activity and physiology.
Sandwich ELISA is a rapid, sensitive, and cost-effective method that uses purified enzyme preparations, adherent, or suspension cell culture extracts for monitoring and assessing kinase activity. It also enables detection of substrate specificity in samples.