Method for extracting and purifying antibody by utilizing magnetic beads

A technology of magnetic beads and antibodies, applied in the field of molecular biology, to achieve the effects of high extraction rate, improved work efficiency, and simple and efficient operation methods

Inactive Publication Date: 2018-07-24
青岛汉德森生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Or the antibody is adsorbed here, b

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  • Method for extracting and purifying antibody by utilizing magnetic beads
  • Method for extracting and purifying antibody by utilizing magnetic beads
  • Method for extracting and purifying antibody by utilizing magnetic beads

Examples

Experimental program
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Embodiment 1

[0059] Example 1: Magnetic Bead Method Sclerotinia-producing Fungus Protein Extraction Kit

[0060] 1.1. Magnetic Bead Method Sclerotia-producing Fungus Protein Extraction Kit includes:

[0061] Step 1: Clamp the sclerotium of the cultured Rhizoctonia solani into a 1.5ml centrifuge tube under sterile conditions, the mass of the sclerotia should not be less than 300mg, use liquid nitrogen to freeze and grind to powder, add 300μL solution Ⅰ , shake and mix;

[0062] Step 2: Centrifuge at 12,000 rpm at 4°C for 1 min, and transfer the supernatant to a new tube;

[0063] Step 3: Add 300 μL of solution II, gently turn up and down 5-6 times;

[0064] Step 6: Add 50 μL of untreated magnetic bead suspension (the magnetic bead is made of nano-magnetic iron oxide, with a diameter of 1.2-2 mm) (purchased from Shanghai Carophen Biomedical Technology Co., Ltd., hydrophilic amino magnetic beads FE03001), After gently turning up and down, let it stand on ice for 10 minutes;

[0065] Ste...

Embodiment 2

[0101] Example 2: Protein Extraction of Samples Using Treated Magnetic Beads

[0102] According to the extraction process described in Example 1, the difference is: before the use of magnetic beads for protein extraction, the purchased magnetic beads are processed, and the processed magnetic beads are used to extract proteins. The practicality and implementation of other methods and reagents The magnetic bead extraction method in Example 1 is the same as the steps.

[0103] The magnetic beads are processed, and the processing method is as follows:

[0104] The magnetic beads were purchased from Shanghai Carboxyphene Biomedical Technology Co., Ltd. (hydrophilic amino magnetic beads FE03001, the average particle size of the nano-magnetic beads is 500 nm), and they were specially used for various coupling reactions. The magnetic beads were treated with 8mol / L oxychloropropane solution and 6mol / L 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride solution soaked the magneti...

Embodiment 3

[0113] Example 3: Determination of protein adsorption capacity using processed magnetic beads

[0114] Use an ultrasonic cell disruptor to disrupt the cell wall of the bacteria to be tested (Staphylococcus aureus), and the specific method of disruption is as follows:

[0115] Bacteria were scraped and made into a suspension with distilled water (10 7 spore / mL), and then place the beaker containing the suspension in the crushing instrument, put the probe of the crushing instrument into the beaker and insert it into the spore suspension, set the crushing time to 10 min in an ice bath environment, and take out the sample for use after cell crushing The protein crude extract was obtained by phenol extraction, and then the crude extract was subjected to magnetic bead adsorption protein detection.

[0116] Add 3-5g magnetic beads of the present invention (the processed magnetic beads in embodiment example 2) to the broken sample liquid, place the beaker on the magnetic stand to c...

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Abstract

The invention discloses a method for extracting and purifying an antibody by utilizing magnetic beads. The method comprises the following steps: preparing hybridoma cell supernatant in a beaker; adding the magnetic beads into the supernatant, wherein the ratio of the volume of the supernatant to the magnetic beads is that 20mg of the magnetic beads correspond to 100ml of the supernatant; putting the beaker on a magnetic frame and adsorbing the antibody by the magnetic beads; after adsorbing for 10min, taking out the magnetic beads; after airing, washing with ethanol for a plurality of times; then eluting the antibody on the magnetic beads with 0.1M glycine with the pH (Potential of Hydrogen) value of 3.0; carrying out elution according to the following number of times of eluting: firstly,eluting the magnetic beads for one time by utilizing 50 microliters of 0.15M NaCl, 20mM Na2HPO4, and a buffering solution with the pH value of 7.0, and discarding an eluting solution; then eluting themagnetic beads by utilizing the 0.1M glycine with the pH value of 3.0 and eluting by utilizing 50 microliters of the glycine each time; collecting an eluting solution to obtain a target antibody solution. The method is simple and rapid to operate.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a kit for extracting proteins from sclerotia-producing fungi by magnetic bead method, and a method for rapidly extracting proteins from sclerotia-producing fungi with strong specificity, in particular, it belongs to a method for purifying antibodies. Background technique [0002] Sclerotia is a dormant body composed of closely intertwined hyphae, and its main function is to resist adverse environments. Common sclerotia-producing fungi include Sclerotinia and Rhizoctonia, which contain a large amount of exopolysaccharides. The fungal exopolysaccharide has the characteristics of high adsorption and high viscosity, which is one of the difficulties in the separation and extraction of high-purity protein from sclerotia-producing fungi. Due to the limitations of genomics itself, scientists have further proposed proteomics research. The separation and purification of prote...

Claims

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Application Information

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IPC IPC(8): C07K1/22C07K16/00
CPCC07K1/22C07K16/00
Inventor 付春辉
Owner 青岛汉德森生物科技有限公司
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