Anchoring method of target biomolecule, expansion microscopic imaging method and application thereof
A technology of biomolecules and labeled molecules, which is applied in the direction of analyzing materials, material excitation analysis, and material analysis through optical means, so as to achieve the effect of facilitating expansion microscopic imaging
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0130] 1. The purpose of the experiment: Using the existing ExM method, it is proved that biomolecules such as lipids cannot be imaged by the existing ExM method.
[0131] 2. Experimental steps:
[0132] Primary hippocampal neurons and HeLa cells were labeled with alkynyl-choline, alkynyl-palmitic acid, alkynyl-farnesol, and the fat-soluble dye DiI, followed by ExM imaging.
[0133] ExM imaging procedure: samples were labeled with 0.1 mg / mL AcX overnight at room temperature, or cross-linked with 0.25% glutaraldehyde for 15 minutes. Prepare a monomer solution (1×PBS, 2M NaCl, 2.5% acrylamide, 0.15% N,N'-methylenebisacrylamide, 8.625% sodium acrylate), and add the newly prepared 10% tetramethylethylene Amine (TEMED) and 10% ammonium persulfate (APS), the cells were first incubated at 4°C for 5 minutes, and then placed in a 37°C incubator to incubate the gel for one hour. The gel was then digested with 8 U proteinase K in digestion buffer (50 mM Tris pH 8.0, 1 mM EDTA, 0.1% Tri...
Embodiment 2
[0137] 1. The purpose of the experiment: to prove that the streptavidin-based click-ExM method can be used to anchor biomolecules such as lipids by means of mass spectrometry, protein gel electrophoresis, and fluorescence imaging.
[0138] 2. Experimental steps:
[0139] 1) Streptavidin and biotin were co-incubated overnight, followed by MALDI mass spectrometry.
[0140] 2) For the proteinase K digestion experiment, the complex of streptavidin and biotin was incubated with proteinase K for different times, and the coomassie brilliant blue imaging of the protein gel was performed.
[0141] 3) For HeLa cell metabolic labeling of alkynyl-choline, respectively click on the organic dye tetramethylrhodamine (TAMRA) or Alexa Fluor 555-labeled streptavidin, after fixation, use methanol, Triton-X100, ethanol / 10% formamide, 10% SDS for treatment.
[0142] 3. Experimental results:
[0143] like Figure 4 shown in a to f, where, Figure 4 a shows the crystal structure of streptavidin....
Embodiment 3
[0145] 1. Experimental purpose: Through the immunofluorescence experiment on tubulin, it is proved that signal amplification can be achieved based on the iterative interaction between streptavidin and biotin trimer.
[0146] 2. Experimental steps:
[0147] Synthetic route of biotin trimer: 10mg biotin-PEG 3 -Amines were placed in 100 μL dimethylformamide, to which 100 μL 1,3,5-benzenetricarboxylic acid tris-(2,3,5,6-tetrafluorophenyl)ester was added ) (concentration is 0.474mg mL -1 ), stirred at room temperature overnight, and the solvent was removed by rotary evaporation. The product was purified by silica gel column (methanol:ethyl acetate from 1:100 to 3:7) to give the product as white solid (7 mg, 68%).
[0148] Its NMR test data are: 1 H NMR (500MHz, CD3OD3)8.45(s,3H),4.84(s,9H),4.50-4.46(m,3H),4.30-4.27(m,3H),3.70-3.56(m,36H),3.51- 3.48(m,6H),3.33-3.29(m,9H),3.19-3.15(m,3H),2.93-2.88(m,3H),2.69(d,J=10.5Hz,3H),2.21-2.16( m,6H), 1.74-1.53(m,12H), 1.44-1.37(m,6H).
...
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