SPR: Protein Kinase Activity Detection Method

Surface plasmon resonance

As one of the most important potential drug targets at present, and a series of drugs targeting protein kinases have been successfully developed, various enzyme activity detection service companies are competing to develop related kinase detection methods and kits for screening and optimizing new ones Compounds are candidates for new drugs. Surface Plasmon Resonance (SPR) method, as a biological analysis technology, is an ideal technology for studying the interaction force between molecules. It is currently commonly used in Biacore, a biological macromolecular interaction analysis system. Some researchers have developed a fast and simple method for detecting protein kinase activity based on SPR technology.

In cell signal transduction, cell cycle regulation and other systems, various protein kinases form a crisscross network, which plays a broad role in cell life activities. Protein kinases mediate a large number of signal transduction in eukaryotic cells and affect the activity of substrate proteins through phosphorylation modification. It also controls many physiological activities in cells, including metabolism, cytoskeleton redistribution, and cell movement. Protein phosphorylation is a very important biological process, which participates in various physiological functions such as growth and development, balance regulation, and internal communication of the nervous and immune system.

At present, it is known that when some protein kinases have point mutations and sequence deletions that cause abnormal activity or expression levels of the kinases, cancers are the largest proportion, and they also include inflammation, diabetes, obesity, autoimmune diseases, neurodegenerative diseases, Cardiovascular diseases and metabolic diseases, etc. Protein kinase has high activity in many malignant tumors, and it is now one of the most important potential drug targets. Protein kinase enzyme activity testing services can help drug screening research. Medicilon provides enzyme activity testing services, such as lipase, xylanase, dismutase, cellulase, hemicellulase, protease, β-glucanase, urease, glucoamylase, phytase, starch Enzyme, pectinase, papain, alpha-amylase preparations, refined pectinase, glucoamylase, lipase, keratinase and other enzyme preparation testing services.

Surface plasmon resonance is a physical optical phenomenon. When a beam of plane light waves enters medium 2 from medium 1, part of the incident light is reflected on the contact interface of medium 1, and the other part is transmitted into the medium. SPR technology is a biosensing analysis and detection technology based on the SPR principle. The real-time sensor map changes due to the change of the refractive index of the medium close to the sensor chip surface. For most molecules, the changes in the SPR angle caused by the molecules of the same mass at the interface are equivalent, so the result of SPR monitoring is actually the change in the quality of the material on the chip surface.

No need to use markers for analysis, so that SPR technology is widely used in the determination of various biological systems. The analysis sample types range from small molecule compounds, peptides, proteins, oligonucleotides, oligosaccharides to lipids, liposomes, and even phages and viruses. With cells, study whether there is binding between molecules and the affinity of the interaction, the speed of binding/dissociation, analyze the binding site and binding sequence, and look for receptors, ligands, substrates, disease targets, etc.

The researchers developed a fast and simple method to determine the activity of protein kinases. A polypeptide substrate with cysteine at one end was fixed on the surface of a gold film. After the protein was phosphorylated, a zinc () compound (biotinylated Zn2L3+) was added. , And finally add streptavidin (SA) to further amplify the signal. SPR imaging technology can also be used to detect the reactions on multiple chips at the same time to analyze the peptide probes fixed on the chip. This method is commonly used to evaluate the degree of protein phosphorylation on the chip surface and is very effective for determining protein kinase activity and inhibitor screening.

Other researchers have invented a fast and effective method involving the use of surface plasmon resonance (hereinafter referred to as SPR) analysis technology to achieve simple analysis of protein kinase activity. More specifically, this method is an application of a novel analysis technique for protein kinase activity and surface plasmon resonance imaging (SPR imaging) that generates images based on SPR analysis of substance interactions. The researchers found a method that, if used with the SPR method, does not require the use of labels, such as fluorescent or radioactive substances. The use of chelating compounds reduces costs and is easy to handle. In addition, due to this use, the method is not affected by the type of phosphorylated amino acid or adjacent amino acid sequence. Moreover, this invention can comprehensively analyze many types of protein kinase signals, which leads to effective distribution of protein kinase dynamics between cells with unknown genes after drug administration or introduction of genes. Therefore, the invention is expected to be applied in the development of genomic drugs, such as analyzing the function of new genes and as a drug research tool.

With the development of sensor technology, SPR technology has the advantages of real-time, fast, no labeling, high sensitivity, and good repeatability, and has been favored by people. It is now mainly used in the following analytical fields: the study of molecular interaction modes; dynamics Constant determination; affinity constant determination and concentration measurement. SPR technology is used in life science research fields: proteomics research, cancer research, new drug development, signal transmission, molecular recognition, immunomodulation, immunoassay, vaccines Development, transient binding, ligand fishing, binding specificity, relationship between structure and function, enzyme reaction, etc. With the continuous development and improvement of SPR instrument and sensor chip technology, combined with the expansion of research in various fields, the application of SPR technology will become more diversified and have broad development prospects.

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