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Preparation method of nano magnetic beads for purifying histidine tagged protein

A nano-magnetic bead and histidine-labeled technology, which is applied in the field of nano-magnetic bead preparation, can solve the problems of complex steps in the synthesis process, difficulty in connecting to the surface of magnetic beads, and high requirements for sample solution, and achieve simple purification and separation , low cost, short time-consuming effect

Active Publication Date: 2019-05-17
南京青柠生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this column purification method has several disadvantages: 1. The requirements for the sample solution are relatively high, and cell debris and impurities must be removed before loading the sample to avoid clogging the column
However, this patented synthesis process has many and complicated steps, and the reaction efficiency of NTA is low (acrylic acid is difficult to react with NTA under the action of EDC and NHS), and it is difficult to connect to the surface of magnetic beads. In addition, the whole process requires many reactants and is expensive , if EDC, NHS, NTA, etc., the reaction cost is high

Method used

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  • Preparation method of nano magnetic beads for purifying histidine tagged protein
  • Preparation method of nano magnetic beads for purifying histidine tagged protein
  • Preparation method of nano magnetic beads for purifying histidine tagged protein

Examples

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Embodiment 1

[0041] This example relates to the preparation and application of a new type of nano-magnetic beads for purifying histidine-tagged proteins.

[0042] The preparation method of magnetic beads comprises the following steps:

[0043] The first step, reactant 0.5 gram sodium alginate, 0.5 gram FeCl 3 ·6H 2 O, 1.5 g NaAc 3H 2 O. 1 milliliter of PEG (MW: 300-6000) and 1 milliliter of deionized water were mixed in 20 milliliters of ethylene glycol solvent, and mechanically stirred at 300 rpm until completely dissolved. Under the conditions of reaction for 25 hours, after cooling to room temperature, the reaction product was moved to a 50 ml centrifuge tube, washed with deionized water, collected with a magnet, and stored in vacuum to obtain black Fe 3 o 4 nanoparticles.

[0044] In the second step, cross-linked sodium alginate is coated on the surface of nanoparticles: 1 g of Fe 3 o 4 Nanoparticles, 1.2 grams of sodium alginate, 0.5 grams of sodium hydroxide, 2 milliliters of ...

Embodiment 2

[0057] This example relates to the preparation and application of a new type of nano-magnetic beads for purifying histidine-tagged proteins.

[0058] The preparation method of magnetic beads comprises the following steps:

[0059] The first step, reactant 0.1 gram sodium alginate, 0.05 gram FeCl 3 ·6H 2 O, 0.05 g NaAc 3H 2 O. 0.1 milliliters of PEG (MW: 300-6000) and 0.1 milliliters of deionized water were mixed in 20 milliliters of ethylene glycol solvents, mechanically stirred at 300 rpm until completely dissolved, then the above solution was transferred to a 50 milliliter reaction kettle, and heated at 100°C Under the conditions of reaction for 48 hours, after cooling to room temperature, the reaction product was moved to a 50 ml centrifuge tube, washed with deionized water, collected with a magnet, and stored in vacuum to obtain black Fe 3 o 4 nanoparticles.

[0060] In the second step, cross-linked sodium alginate is coated on the surface of nanoparticles: 0.5 g of F...

Embodiment 3

[0066] This example relates to the preparation and application of a new type of nano-magnetic beads for purifying histidine-tagged proteins.

[0067] The preparation method of magnetic beads comprises the following steps:

[0068] In the first step, reactant 1 gram of sodium alginate, 1 gram of FeCl 3 ·6H 2 O, 3 g NaAc 3H 2 O. 2 milliliters of PEG (MW: 300~6000) and 2 milliliters of deionized water are mixed in 20 milliliters of ethylene glycol solvents, and after mechanical stirring at 300 rpm until completely dissolved, the above-mentioned solution is transferred to a 50 milliliter reaction kettle and heated at 250° C. Under the conditions of reaction for 2 hours, after cooling to room temperature, the reaction product was transferred to a 50 ml centrifuge tube, washed with deionized water, collected with a magnet, and stored in vacuum to obtain black Fe 3 o 4 nanoparticles.

[0069] In the second step, cross-linked sodium alginate is coated on the surface of nanopartic...

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Abstract

The invention discloses a preparation method of nano magnetic beads for purifying histidine tagged protein. The method comprises the following steps: taking sodium alginate, FeCl3.6H2O, NaAc.3H2O andPEG as raw materials, taking ethylene glycol and deionized water as solvent, reacting through a solvothermal method to generate monodispersed sodium alginate / Fe3O4 nano magnetic beads, subsequently further carrying out an epoxy chloropropane cross-linking reaction and activation on the nano magnetic beads and the newly added sodium alginate, then introducing an iminodiacetic acid (IDA) chelating agent so as to obtain water-stable nano magnetic beads efficiently chelated with metal nickel or cobalt ions; and efficient, rapid, simple and low-cost separation and purification of the histidine tagged protein are realized. The method disclosed by the invention is simple, rapid and stable to operate and low in cost; the synthesized nano magnetic beads are large in specific surface area and stablein a water phase and not prone to agglomeration and precipitation; the purification efficiency of the histidine tagged protein is greatly improved; and the large-batch industrial production of the nano magnetic beads can be realized.

Description

technical field [0001] The invention relates to the technical field of biological materials, in particular to a method for preparing nano magnetic beads used for purifying histidine-tagged proteins. Background technique [0002] At present, the purification methods of histidine-tagged proteins mainly use agarose or dextran microspheres as solid phase carriers, and immobilize the chelating agent iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) on the microspheres through chemical cross-linking. surface, and then fill the column with microspheres to purify histidine-tagged proteins by means of an affinity chromatography column. However, this column purification method has several defects: 1. The requirements for the sample solution are relatively high, and cell debris and impurities must be removed before sample loading to avoid clogging the column. 2. It is necessary to control the sample loading speed to prevent the column from collapsing due to excessive pressure. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J13/14C08L5/04C08K3/22
Inventor 乔秀华
Owner 南京青柠生物科技有限公司
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