Method for separating and purifying sea urchin multifunctional actin

A technology for separation and purification of actin, which is applied in peptide preparation methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of unfavorable protein activity maintenance, complicated operation, and many steps, and achieve simple purification process Ease of operation, simple sample preparation, and the effect of fewer steps

Inactive Publication Date: 2015-03-11
LIAONING OCEAN & FISHERIES SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purification of a certain protein often requires the combination of the above two or more methods, which involves many steps, complicated operation, long time and is not conducive to the maintenance of protein activity.

Method used

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  • Method for separating and purifying sea urchin multifunctional actin
  • Method for separating and purifying sea urchin multifunctional actin
  • Method for separating and purifying sea urchin multifunctional actin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Non-denaturing non-reducing electrophoresis-gel fragmentation and dissolution method for separation and purification of echinococcus urchin MA

[0020] (1) Preparation of body cavity fluid supernatant

[0021] Use a sterilized syringe to penetrate the periosteal membrane of Echinococcus photicus to extract the body cavity fluid, centrifuge (4°C, 3500 rpm, 10 min), and take the supernatant as the body cavity fluid supernatant.

[0022] (2) Non-denaturing and non-reducing dialysis electrophoresis and polyacrylamide gel electrophoresis were performed using the body cavity fluid supernatant as a sample

[0023] ① Prepare Tris-glycine buffer solution (12.0g Tris, 60.0g glycine, 4L ultrapure water), and pre-cool to 4°C.

[0024] ②Put the supernatant of the body cavity fluid into a 10kDa dialysis bag, seal it with a rubber band, then put the 10kDa dialysis bag into a 0.1kDa dialysis bag, fill the gap between the 10kDa dialysis bag and the 0.1kDa dialysis bag with e...

Embodiment 2

[0050] Example 2: Non-denaturing non-reducing electrophoresis-gel fragmentation and dissolution method for separation and purification of Ezoma dung sea urchin MA

[0051] (1) Preparation of body cavity fluid supernatant

[0052] Use a sterilized syringe to pierce the periosteal membrane of Ezoma fecal sea urchin to extract the body cavity fluid, centrifuge (4°C, 3500 rpm, 10 min), and take the supernatant as the body cavity fluid supernatant.

[0053] (2) Non-denaturing and non-reducing dialysis electrophoresis and polyacrylamide gel electrophoresis were performed using the body cavity fluid supernatant as a sample

[0054] Same as step (2) in Example 1, replace the sample with the coelom fluid supernatant of Ezoma dung sea urchin.

[0055] (3) According to the unique color of the MA band after non-denaturing and non-reducing polyacrylamide gel electrophoresis, the gel was cut, and the sea urchin MA was recovered by gel breaking and dissolution technology

[0056] After the...

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Abstract

The invention discloses a method for separating and purifying sea urchin multifunctional actin. The method is characterized by comprising the following steps: (1) taking coelomic fluid of sea urchin, and centrifuging to take the supernatant so as to obtain the coelomic fluid supernatant; (2) taking the coelomic fluid supernatant as a sample to carry out native non-reductive dialysis electrophoresis; (3) carrying out native non-reductive polyacrylamide gel electrophoresis after the dialysis electrophoresis; (4) carrying out gel cutting on the multifunctional actin strip according to the specific color of the sea urchin multifunctional actin strip after the native non-reductive polyacrylamide gel electrophoresis, and recovering the protein in the gel through a gel crushing dissolution and release method so as to obtain the purified sea urchin multifunctional actin. The method has the advantages that (1) the purified sample is simple and rapid to prepare; (2) the reagent and the equipment are simple in requirement, easy to operate, less in step and short in time; (3) the purified sample has high purity and high protein activity.

Description

technical field [0001] The invention relates to a method for separating and purifying proteins, in particular to a method for separating and purifying multifunctional actin in coelomial fluid of sea urchins, and belongs to the technical field of echinoderm physiology. Background technique [0002] Sea urchins belong to the highest group of invertebrates - Echinodermata, and the completion of their physiological activities depends on the participation of many physiological factors. Actin is one of the main components of the cytoskeleton in invertebrates. It is involved in various cellular functions, including cell deformation and contraction, cell activation, and cell division. It is also closely related to immune functions such as damage repair. [0003] Phenoloxidase (PO) is one of the most important immune factors in invertebrates. Through the reaction of oxidizing phenolic substrates, it participates in the limitation and isolation of pathogenic foreign bodies, wound heal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C07K14/435C07K1/26
CPCC12N9/0091C07K14/43504C12N9/0057C12Y116/03001
Inventor 蒋经伟董颖陈仲高杉关晓燕姜北周遵春
Owner LIAONING OCEAN & FISHERIES SCI RES INST
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