Polypeptide specifically binding to trb3 protein, its screening method, identification and use
A specific, selected technology, applied in the field of medicine, can solve the problems of lack of ATP binding sites and catalytic residues, lack of kinase activity, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0033] Example 1 The surface plasmon resonance method is used to screen peptides that bind to TRB3 protein.
[0034]First, the P62 protein is truncated into different polypeptide fragments, and the peptides are synthesized with a peptide solid-phase synthesizer. This process is carried out by Beijing Saibaisheng Gene Co., Ltd.
[0035] EXAMPLES The entire screening process was carried out in a surface plasmon resonance instrument Biacore T200.
[0036] The screening method is as follows:
[0037] 1. The purified protein TRB3 (purchased from RD Company) was coupled to a CM5 chip (purchased from GE Company) through amino groups, the unbound protein was washed away at a flow rate of 10 μL / min, and the surface of the chip was equilibrated for 2 hours.
[0038] 2. Automatically inject 250 μL of different concentrations of polypeptide fragments (200, 50, 12.5, 6.25 nM), and the whole process is carried out at 25°C. The buffer used was HBS-EP buffer (0.01M HEPES, 0.15M NaCl, 3mM ED...
Embodiment 2
[0044] Example 2 The ELISA method verifies the binding of peptides B1, A2 and B3 to protein TRB3.
[0045] The specific operation steps are as follows:
[0046] 1. Dilute human TRB3 protein and bovine serum albumin (BSA) to 10 μg / ml with PBS, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.
[0047] 2. Wash three times with PBS containing 0.1% Tween-20. Coat the plate with 200 μl of blocking solution (10% bovine serum in PBS), and coat at 37°C for 2 hours.
[0048] 3. Pour off the coating solution, add 200 μl of 1 μg / ml polypeptide B1, A2 and B3 solutions, and set up positive control wells, add 200 μl of 1 μg / ml P62 protein solution, and incubate at 37°C for 1 hour.
[0049] 4. Wash five times with PBS containing 0.1% Tween-20. Add 100 μl anti-M13 monoclonal antibody diluted with blocking solution 1:4000 to each well, and incubate at room temperature for 1 h.
[0050] 5. Wash six times with PBS containing 0.1% Tween-20. Prepare substrate chromogenic...
Embodiment 3
[0053] Example 3 The method of competition ELISA verifies that peptides B1, A2 and B3 can compete for the binding of TRB3 to P62 protein.
[0054] The specific operation steps are as follows:
[0055] 1. Dilute human TRB3 protein and bovine serum albumin (BSA) with PBS to 10 μl / ml, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.
[0056] 2. Wash three times with PBS containing 0.1% Tween-20. Coat the plate with 200 μl of blocking solution (10% bovine serum in PBS), and coat at 37°C for 2 hours.
[0057] 3. Pour off the coating solution, add 200 μl of 1 μg / ml P62 protein solution, and incubate at 37°C for 1 hour.
[0058] 4. Wash five times with PBS containing 0.1% Tween-20. Add 100 μl of horseradish catalase-labeled polypeptides B1, A2 and B3 diluted with blocking solution to each well, and incubate at room temperature for 1 h.
[0059] 5. Wash six times with PBS containing 0.1% Tween-20. Prepare substrate chromogenic solution (100mmol / L sodium ace...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More - R&D
- Intellectual Property
- Life Sciences
- Materials
- Tech Scout
- Unparalleled Data Quality
- Higher Quality Content
- 60% Fewer Hallucinations
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com