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A kind of biopolymer cross-linking agent for film and its preparation method and application

A biopolymer and cross-linking agent technology, applied in biochemical equipment and methods, microbial measurement/testing, analytical materials, etc., can solve problems affecting sensitivity, loss, human injury, etc., and achieve the effect of improving detection sensitivity

Active Publication Date: 2018-02-02
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, ultraviolet radiation has a destructive effect on nucleic acid protein molecules, and also has a harmful effect on the human body.
The protein is not firmly fixed on the membrane, especially the nitrocellulose membrane by drying, and will be lost in the subsequent drifting process, which will affect the sensitivity of the detection.

Method used

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  • A kind of biopolymer cross-linking agent for film and its preparation method and application
  • A kind of biopolymer cross-linking agent for film and its preparation method and application
  • A kind of biopolymer cross-linking agent for film and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The preparation of embodiment 1 film biopolymer cross-linking agent

[0032] Take 10mg of arginine, 10mg of tryptophan, 10mg of tyrosine, 1ul Triton-X-100 (commercialized as a liquid) dissolved in 10ml of 0.01% polylysine solution (commercially available) and mix well to obtain a film with biopolymer crosslinkers.

Embodiment 2

[0033] The detection of embodiment 2 RNA molecules

[0034] (1) RNA molecule miR-21 was synthesized by GenePharma Company; a biotin-labeled DNA probe (antisense sequence of miR-21) was synthesized by Shanghai Boshang Biotechnology Co., Ltd.

[0035] (2) Take 10 pmol of the above-mentioned synthetic RNA and DNA probe and carry out liquid phase hybridization according to LHCD (liquid phase hybridization chromogenic method) (X.Li, M.Ni, and Y.Zhang, Methods85(2), 151(2012)) .

[0036] (3) Soak the nylon membrane prepared in Example 1 with a biopolymer cross-linking agent at room temperature for 10 seconds, air-dry or UV cross-link according to professional standard methods.

[0037] (4) Spot 1 μL of the hybridization solution from step (2) on the nylon membrane treated in step (3), and air-dry.

[0038] (5) The next step is to block and add ABC (avidin-biotin complex) signal amplification and subsequent washing of the membrane according to LHCD (liquid phase hybridization color...

Embodiment 3

[0041] The detection of embodiment 3 protein molecules

[0042] (1) The FLAG TAG peptide was synthesized by Gill Biochemical Co., Ltd.

[0043] (2) Soak the nitrocellulose membrane with the biopolymer cross-linking agent prepared in Example 1 at room temperature for 10 seconds, and air-dry.

[0044] (3) Spot 50ng of the protein from step (1) on the membrane treated in step (2), and air-dry or directly spot and air-dry without step (2) treatment.

[0045] (4) Block the nitrocellulose membrane with 10% BSA (bovine serum albumin) at a mass volume ratio for 1 hour at room temperature.

[0046] (5) Hybridize with biotin-labeled rabbit anti-FLAG TAG antibody (purchased from Shanghai Boyao Biological Reagent Co., Ltd.) for 1 hour at room temperature.

[0047] (6) TBST (prepared according to "Molecular Cloning") washed 3 times, 10 minutes each time.

[0048] (7) Amplify the signal with ABC (avidin-biotin complex) and then wash the membrane according to LHCD (liquid phase hybridizat...

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Abstract

The invention relates to a biopolymer crosslinking agent for membranes, a preparation method and application thereof, and belongs to the field of biotechnology. The biopolymer cross-linking agent for membranes comprises the following components in terms of mass percentage: 0.001-0.025% polylysine, 0.05-0.2% arginine, 0.01-1% tryptophan, and 0.01-1% tyrosine %, Triton‑X‑1000.0002~0.005% (V / V), the balance is water with PH=8‑10. The above-mentioned components of the preparation percentage are mixed to obtain a biopolymer crosslinking agent for membranes. The biopolymer cross-linking agent for membranes provided by the invention can directly cross-link nucleic acids and proteins to nucleic acid / protein transfer membranes, eliminating the need for instruments to fix nucleic acids or proteins, and improving the detection sensitivity of nucleic acids / proteins.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a biopolymer crosslinking agent for membranes, a preparation method thereof and its application in the nucleic acid / protein field. Background technique [0002] Blotting technology is a common basic technology for nucleic acid and protein detection. Nucleic acid is represented by Northern blotting, and protein is represented by Western blotting. These two techniques are the most classical and commonly used basic techniques in molecular biology. What they all have in common is the immobilization of biomolecules on commercial membranes. Nucleic acid can be immobilized by high-temperature drying and ultraviolet crosslinking, while protein is mainly dried. Immobilization is extremely important to increase the binding capacity of biomolecules on the membrane to improve detection sensitivity. Ultraviolet cross-linking and high-temperature drying must be realized by specific instruments, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08J7/00C08J3/24C12Q1/6834G01N33/545G01N33/548
Inventor 李湘麒谭毅张弛梁广
Owner WENZHOU MEDICAL UNIV
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