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Fluorescent-labeled cytochrome c immobilization method and application thereof

A cytochrome and fluorescent labeling technology, which is applied in the direction of fluorescence/phosphorescence, material analysis through optical means, and measuring devices, etc., to achieve the effect of wide application range and good economic value

Inactive Publication Date: 2018-06-01
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there are no methods and products using immobilized fluorescently labeled cytochrome c to detect nitric oxide at home and abroad.

Method used

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  • Fluorescent-labeled cytochrome c immobilization method and application thereof
  • Fluorescent-labeled cytochrome c immobilization method and application thereof
  • Fluorescent-labeled cytochrome c immobilization method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Isolation, purification and identification of photosynthetic bacteria cytochrome c

[0035] Cytochrome c was isolated from Rhodopseudomonas palustris ATCC:17001. Centrifuge the fermentation broth of Rhodopseudomonas palustris, collect the cells and resuspend them in Tris-HCL (0.1M, pH 7.4) buffer at 1 / 100 of the volume of the fermentation broth, freeze at -20°C overnight, and thaw at room temperature. Sonicated at 4°C for 10 minutes, refrigerated and centrifuged at 15,000 g for 20 minutes, and the supernatant was taken.

[0036] Equilibrate the DEAE cellulose chromatography column with Tris-HCL (0.01M, pH7.4) buffer, rinse with 10 times column volume Tris-HCL (0.01M, pH7.4) buffer after loading, and then use low Wash with ionic strength NaCl solution (0.02M) until no protein components flow out, and finally add high ionic strength NaCL (0.2M), collect and combine eluents, and concentrate.

[0037] The concentrated solution was loaded onto a SephadexG-100 mo...

Embodiment 2

[0038] Example 2: Fluorescence labeling of cytochrome c

[0039] Cytochrome c was treated with 0.1M NaHCO 3 Dilute the solution to 500 μg / mL, dissolve hexachlorofluorescein succinimide ester with DMSO to 20 mM, take 5 μL and add it to the above cytochrome c solution, mix and react at room temperature for 1 h, buffer with 0.1M Tris-HCL pH7.4 The dialysate was dialyzed until the dialysate was free of fluorescent substances.

Embodiment 3

[0040] Example 3: Immobilization of fluorescently labeled cytochrome c

[0041] Take 2g of aluminum isopropoxide, add 44ml of boiling water, stir at 80°C for 45min, add 1.2mL of 1M HCl, boil for 1h, cool to 4°C, and prepare aluminum glue mother liquor.

[0042] Mix the fluorescently labeled cytochrome c and the aluminum glue mother solution at a volume ratio of 1:1, mix well and let stand at 4°C for 1 hour, spread evenly on glass beads, and store in a refrigerator at 4°C overnight for later use.

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Abstract

The application relates to a fluorescent-labeled cytochrome c immobilization method and application thereof, and belongs to the field of biological detection. The method comprises the following steps:(1) extracting purified cytochrome c from microorganisms; (2) making the cytochrome c react with fluorescein to prepare a fluorescent-labeled cytochrome c, and removing free fluorescein; (3) preparing an alumina gel mother solution with aluminum isopropoxide; (4) uniformly mixing products obtained in the step (2) and the step (3), and coating a solid-phase carrier. An immobilized fluorescent-labeled cytochrome c prepared by the method has quick response to NO in the solution, can be recycled, and has a better application prospect.

Description

[0001] This application is a divisional application with the application number 2017101669312, the filing date is March 20, 2017, and the title of the invention is "an immobilized fluorescently labeled cytochrome c, its preparation method and application". technical field [0002] The invention belongs to the field of biological detection, and in particular relates to a method for immobilizing fluorescently labeled cytochrome c and the use of the immobilized fluorescently labeled cytochrome c in the detection of biological samples. Background technique [0003] Nitric oxide (NO) is a messenger molecule and effector molecule with various biological activities. It plays an important role in regulating the cardiovascular and cerebrovascular systems, digestive system, and nervous system. Recent studies have shown that NO can also play a non-specific host defense role. Mediate pathological processes such as tissue damage and apoptosis, and then cause inflammatory reactions and aut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6439
Inventor 唐乾曹洪玉王立皓郑学仿
Owner DALIAN UNIV
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