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Method for preparing porphyra yezoensis R-phycoerythrin fluorescence probe

A phycoerythrin and fluorescent probe technology, which is applied in the field of preparing phycobiliprotein fluorescent probes, can solve the problems of high purity requirements of phycoerythrin and monoclonal antibodies, difficult preparation process of fluorescent probes, etc., and achieves high detection effect. , low cost, low cost effect

Inactive Publication Date: 2010-06-23
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty in the preparation of fluorescent probes and the high purity requirements of the phycoerythrin and monoclonal antibodies used, the phycoerythrin fluorescent markers used by domestic scientific research and medical institutions are completely dependent on imports, which limits their expansion. application bottleneck

Method used

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  • Method for preparing porphyra yezoensis R-phycoerythrin fluorescence probe
  • Method for preparing porphyra yezoensis R-phycoerythrin fluorescence probe
  • Method for preparing porphyra yezoensis R-phycoerythrin fluorescence probe

Examples

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Embodiment 1

[0022] The materials used in this example are Porphyra cerevisiae R-phycoerythrin (R-PE) and goat anti-rabbit antibody. The company purchased the goat anti-rabbit antibody from Wuhan Huamei Bioengineering Co., Ltd.

[0023] (1), carry out R-phycoerythrin with 3-(2-pyridine dimercapto) propionate n-hydroxysuccinimide ester (SPDP) and R-phycoerythrin (R-PE) molar ratio is 40:1 (R-PE) derivatization, shaking at 50 rpm in the dark for 2 hours on a shaker, and then using a Sephadex G-25 desalting column to remove unreacted 3-(2-pyridyldimercapto)propionic acid n-hydroxysuccinate Imide ester (SPDP), collection of derivatized R-phycoerythrin (R-PE);

[0024] (2) The molar ratio of dithiothreitol (DTT) to goat anti-rabbit antibody was 500:1 to carry out the thiolation of the goat anti-rabbit antibody. At 20° C., the shaker was shaken at 50 rpm for 1 hour, Use Sephadex G-25 desalting column to remove unreacted dithiothreitol (DTT), and collect thiolated goat anti-rabbit antibody;

...

Embodiment 2

[0028] The application steps of Porphyra zebra R-phycoerythrin fluorescent probe in the detection of liver cancer SMCC-7721 cells are as follows:

[0029] 1. Take an ordinary clean cover glass and soak it in 70% ethanol for 5 minutes or longer, dry it in a sterile ultra-clean bench, wash it three times with 0.9% sodium chloride solution, and then wash it once with cell culture medium. The coverslips were placed in a six-well plate, seeded into liver cancer SMCC-7721 cells and cultured overnight.

[0030] 2. Aspirate the culture medium in the six-well plate, add 1ml of fixative solution, and fix overnight at 4°C.

[0031] 3. Remove the fixative, wash 3 times with washing solution, 3-5 minutes each time, and suck up the liquid.

[0032] 4. Block with blocking solution for 60 minutes.

[0033] 5. Remove the blocking solution, add diluted rabbit anti-actin polyclonal antibody (1:400) for 60 minutes.

[0034] 6. Remove the primary antibody and wash 3-5 times with washing solutio...

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Abstract

The invention relates to a method for preparing a porphyra yezoensis R-phycoerythrin fluorescence probe, which is characterized in that the method carries out the following steps at the temperature of 20 DEG C: firstly, deriving R-phycoerythrin in a mole ratio of 40 to 1 of 3 - (2 - pyridine 2-mercapto) propionic acid n-hydroxysuccinimide ester and R-phycoerythrin, 2 hours shaker dark slow-speed oscillating for 2 hours, and using Sephadex G-25 desalting columns to collect the derived R-phycoerythrin; then carrying out the sulfhydrylation of an antibody in a mole ratio of 500 to 1 of dithiothreitol and the antibody, shaker slow-speed oscillating for 1 hour, and using the Sephadex G-25 desalting columns to collect the sulfhydrylation antibody; finally, taking the derived R-phycoerythrin according to the ratio of the mass ratio of 3 to 5 of the R-phycoerythrin before derivation and the antibody to mix with the sulfhydrylation antibody, shaker slow-speed oscillating for 12 hours, adding 100muml N-ethyl maleimide for reacting for 30 minutes, separating and purifying by a Sephacryl S-300HR chromatographic column, and using an eluant of 50mmol / L phosphate buffer and 150 mmol / L sodium chloride with a pH value of 7.0 to collect a separated cross-linked product, namely a porphyra yezoensis R-phycoerythrin fluorescence probe.

Description

Technical field: [0001] The invention relates to a method for preparing a phycobiliprotein fluorescent probe, in particular to a method for preparing a Porphyra variegata R-phycoerythrin fluorescent probe. Background technique: [0002] Phycobiliprotein is a good pure natural pigment with bright colors, which can be used as food pigment, cosmetics, additives, etc. At the same time, phycobiliprotein is also a new type of fluorescent substance, including phycoerythrin, phycocyanin and allophycocyanin Blue proteins, etc., can be used as antibody fluorescent labeling substances instead of synthetic fluorescent substances. As fluorescent tracers in living organisms, they are being widely used in clinical diagnosis, immune labeling and other fields. In particular, R-phycoerythrin (R-PE) can avoid most biological environments because of its extremely high extinction coefficient and quantum yield, large Stokes (STOKES) shift, and its yellow-orange characteristic fluorescence. The d...

Claims

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Application Information

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IPC IPC(8): G01N33/52G01N21/64
Inventor 何培民蔡春尔周铭汪卿柳俊秀李春霞
Owner SHANGHAI OCEAN UNIV
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