Quantitative evaluation of phosphorylation of hott

Quantitative evaluation of protein phosphorylation

protein phosphorylation is a key step in regulating cellular signals. Using infrared imaging technology, an artificial cultured cell array pattern that can quantify protein phosphorylation has been developed, and can be used to measure the 50% inhibition concentration of pharmacology

abnormal phosphorylation plays a role in many diseases, such as cancer, diabetes induced retinopathy, rheumatoid arthritis and Parkinson's disease. Therefore, many new drugs under development aim at inhibiting protein kinases. Developing this drug is a time-consuming and expensive process. There are many ways to screen kinase inhibitors. Although biochemical test can easily adapt to high-channel screening, it often produces a large proportion of wrong results because this test does not raise problems such as the ability of drugs to enter cells, drug toxicity, or post biological reproduction problems caused by the characteristics of in vitro biological test

li cor developed a new test method based on fluorescence immunocytochemistry, called in cell western. This assay method is carried out on a plate with 96 or 384 holes, and does not require cell extraction. In particular, they pioneered an economical cell tissue culture method through 3D printing processes such as FDM. The artificially cultured cells on the microtiter plate are treated with candidate drugs, and then the required protein kinase signal pathway is introduced. The cells are fixed and can be infiltrated by standard immunocytochemical methods. Primary antibodies derived from two different species were introduced at the same time. A primary antibody targets pan target proteins or "housekeeping proteins and filtration, catalysts and coatings in manufacturing". Another antibody targets the target protein of the ammonium phosphate enzyme. Then, the two primary antibodies were calibrated with Alexa fluor 680 and irdye 800 (two fluorescent dyes) to form secondary antibodies, which were introduced into the system. The total fluorescence emitted from each micro titrated sample cell can be quantitatively measured at 700nm and 800nm wavelengths by Odyssey infrared imager (Fig. 1)

results and discussion

first, the specificity of the antibody used in the in cell Western test and the absence of spurious bands in the previously mentioned Western two-color imprint should be checked

in order to illustrate the utility of in cell Western test in determining IC50 (inhibitory concentration) value in drug characterization, the increase in the number of pd168393 was monitored, which responded to the drug's inhibition of extracellular signal regulated kinase (ERK) phosphorylation. Pd168393 is a known epidermal growth factor receptor (EGFR) and a member of the extracellular signal regulated kinase (ERK) command level. Pd168393, which is getting denser and denser, was used to pretreat the 384 well artificial A431 cell test disk, whether it is nonlinear or at what level, and the epidermal growth factor (EGF) solution of 100 ng/ml was used for stimulation. The cells are fixed, soaked with detergent and easy to be immunostained

the total amount of extracellular signal regulated kinase (ERK) was obtained by combined analysis of Rabbit anti ERK and irdye800 CW labeled Sheep anti rabbit antibodies. The amount of phosphorous ERK was calculated by the amount of phosphorous mouse anti ERK and Alexa flow 680 labeled Sheep anti mouse. In the presence of EGF and the absence of pd168393, the phosphorylation of extracellular signal regulated kinase (ERK) increased substantially. A related decrease in the dose of phosphorylation was observed depending on the dose of pd168393 added to the sample. The phosphorylation of ERK treated according to the standardization of the total amount of ERK ensures the accurate and repeatable quantitative determination of the utility of pd168393 and the determination of the 50% inhibitory concentration of the drug. In cell western can obtain the accurate IC50 value of pd168393, which is consistent with the IC50 value described in the literature

summary

in cell Western assay relies on two-color infrared detection; Accurate and highly repeatable experimental data can be obtained; Moreover, compared with Western blotting, it does not require intensive work, and there is no potential illusion caused by cell extraction. It is particularly important to avoid cell extraction, because some phosphorus containing proteins are unstable, and dephosphorylation during processing will lead to wrong results. Unlike the chemiluminescence method based on enzyme substrate chemical reaction, infrared fluorescence detection is a direct quantitative method with a linear range of more than 4000 fold. In cell Western detection can accurately determine the effect of drugs on protein kinase targets in the form of convenient microtiter plates without cell extraction and electrophoretic separation: quantitative evaluation. Facts will prove that this detection method is very useful in testing the efficacy of kinase inhibitory drugs. (end)