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Protein extraction and enrichment method for silk proteomics

A proteomics and protein technology, applied in the field of cultural relics detection, can solve the problems of studying the characteristics of silk at the molecular level, without any research on changes, etc., to achieve the effect of convenient cleaning, avoiding protein loss, and wide range of extraction

Pending Publication Date: 2021-07-02
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on silk cultural relics is still limited to the characterization of secondary structures such as scanning and infrared, and it is difficult to study the characteristics of silk at the molecular level. At present, the best method for identifying protein fibers is ELISA, but ELISA can only distinguish protein fibers, but it does not Their protein and peptide composition, changes in the aging process have not been studied, so a more detailed research method for trace proteins needs to be developed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: (enrichment and extraction of silk fibroin powder)

[0035] 1) Take 4 mg of silk fibroin powder in a centrifuge tube, add 2 mL of lysate to it, react at room temperature for 2 hours, and add 2 mL of ammonium bicarbonate solution.

[0036] 2) Take 1mL silk solution from step 1) and add it to a 2mL centrifuge tube, put it into a constant temperature mixer, heat at 60°C for 30 minutes, 1000 rpm.

[0037] 3) Remove the test tube in step 2) from the constant temperature mixer, put it on the shelf on the laboratory bench, and let it cool to room temperature. Add 100 μL of iodoacetamide (IAA) stock solution to the tube to alkylate the reduced disulfide, and incubate in dark box at room temperature for 30 minutes.

[0038] 4) Add 50 μL of dithiothreitol (DTT) slurry to the centrifuge tube in step 3), and incubate on a laboratory bench for 15 minutes at room temperature.

[0039] 5) Preheat the thermostatic mixer to 24°C, take 20 μL of the mixture in step 4), and di...

Embodiment 2

[0044] Example 2: (Extraction and enrichment of all proteins in silkworm cocoons)

[0045] 1) Take 6 mg of shredded silkworm cocoons, add 2 mL of lysate to it in a centrifuge tube, react at room temperature for 2 hours, and add 2 mL of ammonium bicarbonate solution.

[0046] 2) Take 1mL silk solution from step 1) and add it to a 2mL centrifuge tube, put it into a constant temperature mixer, heat at 60°C for 30 minutes, 1000 rpm.

[0047] 3) Remove the test tube in step 2) from the constant temperature mixer, put it on the shelf on the laboratory bench, and let it cool to room temperature. Add 100 μL of iodoacetamide (IAA) stock solution to the tube to alkylate the reduced disulfide, and incubate in dark box at room temperature for 30 minutes.

[0048] 4) Add 50 μL of dithiothreitol (DTT) slurry to the centrifuge tube in step 3), and incubate on a laboratory bench for 15 minutes at room temperature in the dark.

[0049] 5) Preheat the constant temperature mixer to 24°C, take ...

Embodiment 3

[0055]1) Take 1 mg of silk cultural relics in a centrifuge tube, add 0.5 mL of lysate to it, react at room temperature for 2 hours, and add 0.5 mL of ammonium bicarbonate solution.

[0056] 2) Take 1mL silk solution from step 1) and add it to a 2mL centrifuge tube, put it into a constant temperature mixer, heat at 60°C for 30 minutes, 1000 rpm.

[0057] 3) Remove the test tube in step 2) from the constant temperature mixer, put it on the shelf on the laboratory bench, and let it cool to room temperature. Add 100 μL of iodoacetamide (IAA) stock solution to the tube to alkylate the reduced disulfide, and incubate in dark box at room temperature for 30 minutes.

[0058] 4) Add 50 μL of dithiothreitol (DTT) slurry to the centrifuge tube in step 3), and incubate on a laboratory bench for 15 minutes at room temperature.

[0059] 5) Preheat the constant temperature mixer to 24°C, take 25 μL of the mixture in step 4), and dilute to a final volume of 96 μL. Add 200 μg of prepared par...

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Abstract

The invention relates to the technical field of cultural relic detection, and discloses a protein extraction and enrichment method for silk proteomics, the method comprises the following steps: dissolving silk protein, enriching the silk protein on magnetic beads under the combined action of carboxyl salt modified paramagnetic beads and an organic solvent, and extracting the protein from the solution through magnetic separation. The magnetic beads belong to a solid phase and are fixed by the magnetic field so that the protein is simply and conveniently washed and purified, and the purification effect is good.

Description

technical field [0001] The invention relates to the technical field of cultural relic detection, in particular to a protein extraction and enrichment method for silk proteomics. Background technique [0002] Silk has a pivotal position in Chinese history. The Silk Road is a road of cultural exchange starting from the Yellow River and Yangtze River basins in ancient times, connecting North Africa and Europe via India, Central Asia and West Asia, and using silk trade as the main medium. Silk played an important role as a link in ancient Chinese cultural exchanges and was an important part of Chinese traditional culture. During the archaeological process, various unearthed fabrics also told all scientists about the splendid civilization of China. However, silk cultural relics are different from stone tools and bronze tools. As an organic substance, silk is difficult to preserve intact during a long-term burial process. The longer the time, the fewer traces remain. The resear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
CPCC07K1/14
Inventor 王秉周杰李津彭志勤万军民
Owner ZHEJIANG SCI-TECH UNIV
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