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Rasal2 protein 123-site phosphorylation specific antibody, preparation method and ELISA detection kit

A detection kit and phosphorylation technology, applied in chemical instruments and methods, immunoglobulins, anti-enzyme immunoglobulins, etc., can solve the problems that there is no phosphorylated Rasal2 detection antibody, and achieve inhibition of tumor occurrence and high purity , the effect of promoting drug resistance

Inactive Publication Date: 2021-12-17
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there is no detection antibody for phosphorylated Rasal2 as a marker of tumorigenesis and development

Method used

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  • Rasal2 protein 123-site phosphorylation specific antibody, preparation method and ELISA detection kit
  • Rasal2 protein 123-site phosphorylation specific antibody, preparation method and ELISA detection kit
  • Rasal2 protein 123-site phosphorylation specific antibody, preparation method and ELISA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of Ser123 Phosphorylated Rasal2 Complete Antigen

[0048] ① Design the antigenic peptide phosphorylated at S123 site, such as figure 1 As shown, at the same time, according to the amino acid characteristics of the polypeptide sequence, cysteine ​​(Cys) is added at the front end or the end so that the antigen peptide can be coupled with hemocyanin;

[0049] ② Prepare hemocyanin solution, dissolve hemocyanin into PBS, so that the final concentration is 2mg / ml;

[0050] ③ Mix the hemocyanin solution with the Sulfo-SMCC solution, and incubate at room temperature for 4 hours;

[0051] ④ Dialyze with PBS to remove free SMCC, and calculate the protein concentration by the Broadford method;

[0052] ⑤ Mix the synthesized antigen peptide phosphorylated at S123 with hemocyanin-SMCC solution, and incubate at room temperature for 4 hours to prepare Rasal2 phosphorylated antigen at position 123;

Embodiment 2

[0053] Example 2 Preparation of Rasal2 Phosphorylated Antibody

[0054] ① Phosphorylated complete antigen at the 123rd position of Rasal2 was added with an equal amount of complete Freund's adjuvant and emulsified;

[0055] ② Inject the emulsified antigen into the back of New Zealand rabbits at multiple points intradermally, each dose of 0.5mg / kg per rabbit (basic immunity);

[0056] ③ After 20 days of basic immunization, fully mix and emulsify the above antigen with an equal volume of Freund's incomplete adjuvant;

[0057] ④ Inject the emulsified antigen into the back of New Zealand rabbits at multiple points intradermally for the first booster immunization;

[0058] ⑤ After 20 days, perform the second booster immunization according to the operation steps in ④;

[0059] ⑥ After 14 days of the second booster immunization, the immunized animals were sacrificed and blood was collected. Place the serum in an ice-water bath for 4 hours, centrifuge at 5000 rpm for 10 minutes, an...

Embodiment 3

[0061] Example 3 p-Rasal2 (Ser123) antibody specific identification

[0062] Construct the Rasal2 S123A mutant, Myc-Rasal2-S123A, and transfer the recombinant vectors Myc-Rasal2-S123A and Myc-Rasal2-WT into 293T cells respectively, then add glucose starvation for 4 hours, and carry out co-immunoprecipitation through M2-Beads, To identify whether the p-Rasal2 antibody can recognize two differently expressed proteins, so as to determine the specificity of the antibody;

[0063] The result is as figure 2 As shown, where WT: 293T cells transfected with Myc-Rasal2-WT expression vector, S123A: 293T cells transformed with point mutation Myc-Rasal2-S123A;

[0064] The experimental results showed that the obtained anti-p-Rasal2 (Ser123) antibody could specifically recognize the phosphorylation at position 123 of Rasal2, and glucose starvation could significantly increase the phosphorylation of Rasal2 (Ser123).

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a Rasal2 123-site phosphorylated antibody, a preparation method thereof, an ELISA detection kit for detecting Rasal2 123-site phosphorylation and a detection method. The preparation method comprises the following steps: synthesizing an antigen peptide of which S123 site is phosphorylated; coupling the antigen peptide with hemocyanin to obtain an immunizing antigen, then immunizing an animal by using the antigen and collecting antiserum; and identifying and purifying the antiserum to obtain the Rasal2 123-site phosphorylation specific antibody. The ELISA detection kit is obtained by coating an ELISA plate with the Rasal2 123-site phosphorylation specific antibody and assisting with other detection materials. The ELISA detection kit can be used for identifying a 123-th phosphorylation content of Rasal2 protein in a tissue sample, and is simple and convenient to operate, high in specificity, high in sensitivity and easy to standardize.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation of an antibody for phosphorylation at position 123 of Rasal2 and an ELISA detection kit for detecting phosphorylation at position 123 of Rasal2. Background technique [0002] The prior art discloses that Rasal2, as a member of the RasGAP family, inhibits the activity of Ras mainly by promoting the hydrolysis of Ras-GTP into GDP, and thus can inhibit the occurrence and development of tumors in most tumors. Studies have shown that in breast cancer, Rasal2 has two different modes of action, promoting and inhibiting tumor growth. Rasal2 is low expressed in luminal B breast cancer and inhibits tumor growth and invasion by inhibiting Ras activity. However, in triple-negative breast cancer, Rasal2 is overexpressed and interacts with ARHGAP24 to activate RAC1 to promote cell invasion and migration. [0003] Studies have disclosed that the overactivation of Ras is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C07K16/44C07K16/06G01N33/573G01N33/574G01N33/543
CPCC07K16/40C07K16/44C07K16/06G01N33/573G01N33/57484G01N33/54393G01N2333/914
Inventor 钱忠明包勇
Owner FUDAN UNIV
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