Preparation method of protein thermal stability detection chip, detection method and kit
A thermal stability and detection chip technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., to achieve high throughput, true and reliable data, and stable products
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Embodiment 1
[0059] Example 1 A novel coronavirus drug target protein thermostability detection chip
[0060] Principle and application of the protein stability detection chip in this example figure 1 .
[0061] The specially-made protein thermal stability detection chip and kit contain the pure product of the target protein of the new coronavirus in the orifice plate, and the drug molecules are diluted with the sample diluent and added to different areas of the reaction plate.
[0062] The sample diluent is: 0.01M phosphate buffer (sterile filtered) at pH=7.0, and 0.01% sodium azide signal enhancer.
[0063] The fixed parameters of the fluorescent quantitative PCR instrument are used to measure, and the thermal stability curve of the target protein with or without drug molecules is obtained, and the kit is used to judge whether molecular interactions occur.
[0064] For the distribution of protein species and the design of the chip used in the protein thermostability detection chip, see...
Embodiment 2
[0074] Example 2 A kit for screening new coronavirus target drugs
[0075] Including: the protein thermal stability detection chip in Example 1, sample diluent, positive control substance and negative control substance.
[0076] The sample diluent is: 0.01M phosphate buffer (sterile filtered) with pH=7.0, 0.01% sodium azide as a signal enhancer and preservative, and 5 μM fluorescent dye.
[0077] In the early research and development process of this kit, it was found that the fluorescent signal concentration was not strong, and the use of high-concentration proteins would bring about cost increases. In addition, some small molecule drugs also have autofluorescence, which will bring high background. At the same time, the reagents The long-term preservation of the box also requires preservatives. In order to solve the above problems, we tested a variety of chemical molecular additives and tested their performance. The test results are as follows: image 3 shown. The results sh...
Embodiment 3
[0081] Example 3 Target Protein Standard Thermal Stability Curve Quality Control
[0082] The standard thermal stability curve quality control was performed on the protein prepared in Example 1, and 10 μL of protein sample dilution was added to the reaction wells of each protein, with a final volume of 20 μL.
[0083] starting program:
[0084] The quality control chart of all proteins in the protein chip is as follows: Figure 4 , Figure 5 As shown, the normal melting curve of each protein when no drug is added, among which (NFkB1)P50 protein has been tested for four protein concentrations. According to the test results of the protein concentration of the kit, this kit uses NFκB1(P50) protein The final concentration is 0.045 μg / μL, and the concentration of other proteins in this kit is 0.09 μg / μL for experiments.
[0085] The procedure for on-board testing is as follows:
[0086] starting program:
[0087] Step 1, temperature 37°C, time 2 minutes;
[0088] Variable tem...
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