Method for screening combined blocking agent among Bcl-2 family members by utilizing HTRF one-step method

A technology of bcl-2 and blocking agents, which is applied in the direction of material inspection products, fluorescence/phosphorescence, biological testing, etc., can solve problems such as experimental errors, inability to quantify, and long incubation time of antibodies, so as to avoid false positives and false negatives, Save experimental workload and achieve long-lasting fluorescence effects

Inactive Publication Date: 2019-05-28
浠思(上海)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The antibody incubation time of Co-IP is too long, and the subsequent WB operation also takes a long time. The whole cycle takes 2-3 days, and it can only be used for qualitative but not quantitative, and high-throughput screening cannot be performed in drug screening.
As a traditional method, ELISA has some shortcomings that are difficult to overcome, such as: 1) There are many experimental steps, it takes a long time, and it needs to go through multiple washings, adding samples, color development, etc., and it takes at least one day; 2) It cannot reach high-pass 3) The coated protein may shield some protein sites, resulting in missing positive results, that is, false negative results; 4) Due to many steps, it is easy to cause experimental errors, leading to poor repeatability, unstable

Method used

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  • Method for screening combined blocking agent among Bcl-2 family members by utilizing HTRF one-step method
  • Method for screening combined blocking agent among Bcl-2 family members by utilizing HTRF one-step method
  • Method for screening combined blocking agent among Bcl-2 family members by utilizing HTRF one-step method

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Experimental program
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Effect test

Embodiment 1

[0042] 1) For protein preparation, MCL1 was serially diluted 3 times from 200nM to obtain different concentrations from 200nM to 0nM; BAK was serially diluted 3 times from 200nM to obtain different concentrations from 200nM to 0nM;

[0043] 2) Add 5ul each of the prepared proteins into a 384-well detection plate, incubate at room temperature for 15 minutes, add the tag antibody coupled to Donor and Acceptor (or add 10ul after equal volume mixing), incubate for 2 hours and read;

[0044] According to the principle of HTRF method, detect the fluorescence ratio of 665nm / 620nm, according to this ratio, judge the binding of protein, the higher the signal, the more binding, see the results figure 2 .

Embodiment 2

[0046] 1) For positive compounds, compound Selleck A-1210477 and APExBIO S63845 were serially diluted, starting from 3000nM, and diluted 3 times; at the same time, for negative compounds, compound Selleck ABT-737 was diluted 3 times starting from 3000nM;

[0047] 2) Prepare the protein, and perform subsequent blocking agent screening according to the appropriate protein concentration in Example 1, MCL1 is 4nM, and BAK is 8nM;

[0048] 3) The prepared blocking agent and protein were added to the 384-well detection plate in sequence, and after incubating at room temperature for 15 minutes, the tag antibody coupled with Donor and Acceptor was added (or 10ul after equal volume mixing), and incubated for 2 hours for reading;

[0049] According to the principle of HTRF method, detect the fluorescence ratio of 665nm / 620nm, according to this ratio, judge the binding of protein, the higher the signal, the more binding, see the results image 3 .

[0050] Depend on figure 1 , 2, 3 It ...

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Abstract

The invention discloses a method for screening a combined blocking agent among Bcl-2 family members by utilizing an HTRF one-step method, and belongs to the field of blocking agent screening methods.The method for screening the blocking agent combined between Bcl-2 family members (such as Mcl1/ BAK) by utilizing the HTRF one-step method comprises the following steps: configuring constructed Mcl1and BAK proteins with different labels, and screening a sample; sequentially adding the Mcl1 and the BAK proteins of the sample to be screened into a microporous plate, wherein the two corresponding anti-label antibodies are respectively coupled with Donor and Acceptor; incubating at room temperature, and detecting fluorescence by using a microplate reader, wherein the ratio of 665nm/ 620nm is original data; according to the method, screening the combined blocking agent among Bcl-2 family members (such as Mcl1/BAK) at the biochemical level can be achieved, and constructing cells is not needed,and the method is more direct and quicker.

Description

technical field [0001] The invention belongs to the field of blocking agent screening methods, and in particular relates to a method for screening Bcl-2 family blocking agents by using HTRF one-step method. Background technique [0002] Bcl-2 family proteins play a very important role as key regulatory molecules in the apoptosis signaling pathway. The Bcl-2 family includes two types of members, pro-apoptotic proteins and anti-apoptotic proteins, and the two types of members jointly regulate the process of apoptosis by regulating the permeability of the mitochondrial outer membrane. [0003] According to the classic classification method, the Bcl-2 family can be divided into three subfamilies: 1. Pro-survival proteins, including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1 and so on. These proteins inhibit the occurrence of apoptosis by directly interacting with pro-apoptotic proteins. 2. Executive proteins, including Bax and Bak, both of which are considered to be directly involved in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64
Inventor 王心媛
Owner 浠思(上海)生物技术有限公司
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