Antibody of cytochrome b5 type B and application thereof in purifying mitochondria

A mitochondrial, antigen-antibody technology, applied in anti-animal/human immunoglobulins, chemical instruments and methods, carrier binding/immobilization peptides, etc. The effect of high collection efficiency and high purity

Inactive Publication Date: 2012-12-19
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Again, the authors did not systematically examine at what scale and cell number level, the immunomagnetic bead method can produce high-quality and high-yield mitochondria

Method used

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  • Antibody of cytochrome b5 type B and application thereof in purifying mitochondria
  • Antibody of cytochrome b5 type B and application thereof in purifying mitochondria
  • Antibody of cytochrome b5 type B and application thereof in purifying mitochondria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of CYB5B Immunogen

[0044] 1. CYB5B gene cloning

[0045] Design PCR primers according to the published gene sequence of human CYB5B (gi:83921613):

[0046] CYB5B 5'forward primer: 5'CCG GAA TTC ATG TCC GGT TCA ATG GCG 3'

[0047] CYB5B 3' reverse primer: 5' CCG CTC GAG TTT TGA AGG GTC CTT GCT 3'

[0048] Using the human liver cDNA library as a template, the 1-345 nucleic acid sequence of the CYB5B gene (SEQ ID NO: 2) was amplified by PCR. PCR parameters: 95°C for 5min; 94°C for 30s, 54°C for 30s, 72°C for 1min, a total of 40 cycles; After extension at 72°C for 10 min).

[0049] 2. Subcloning of CYB5B gene fragment into pET32a vector

[0050] The CYB5B fragment obtained by the above PCR and the pET32a plasmid were respectively digested with EcoR I and Xho I at 37° C. for 8 hours, and the effect of the enzyme digestion was detected by agarose gel electrophoresis, and then the agarose gel DNA recovery kit (TianGEN Company) was used. Purify the ...

Embodiment 2

[0053] Example 2 Preparation of anti-human CYB5B polyclonal antibody

[0054] 1. Preparation and detection of CYB5B polyclonal antibody

[0055] Take 0.5mL (750ug) of CYB5B recombinant protein and mix it well with an equal volume of Freund's complete adjuvant, and take 1 New Zealand big-eared white rabbit for multiple subcutaneous injections in the back and groin. Two weeks later, booster immunization was started. The dose of booster immunization was 500 μg CYB5B recombinant protein, which was fully mixed with an equal volume of Freund's incomplete adjuvant and injected subcutaneously, once every 2 weeks. Blood was collected 7 days after the fourth booster immunization, and serum was separated.

[0056] Titre the antiserum in the blood. Add 100 μl (0.1ng) of CYB5B recombinant protein to a 96-well plate, cover the 96-well plate with parafilm, and store in a 4-degree refrigerator overnight. Wash three times with TPBS, add 200 μl of TPBS, let stand for 5 minutes, shake off the...

Embodiment 3

[0062] Example 3 Preparation of Mitochondria

[0063] 1. Connection of magnetic beads and antibodies

[0064] Every 100 μl of Dynabeads Protein A magnetic beads (Invitrogen) equilibrated with 0.1 M phosphate buffer at pH 8 was mixed with 25 μg of purified anti-CYB5B antibody on a rotary mixer for 20 minutes at room temperature. Use the magnetic stand to attach the beads and remove the liquid. Wash 3 times with pH 8 0.1M phosphate buffer.

[0065] 2. Enrichment and purity detection of mitochondria

[0066] collect 1x10 7 For each BGC823 cell, wash the cells twice with PBS, resuspend the cells with mitochondrial homogenate, and homogenize 40-60 times on ice. Collect the cell homogenate solution, centrifuge at 500g for 5 minutes, collect the supernatant and put it on ice, resuspend the pellet with mitochondrial homogenate solution and homogenize for 40-60 times, collect the supernatant and combine. After the combined supernatant was centrifuged at 1000g for 10 minutes to rem...

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Abstract

The invention relates to a specific polyclonal antibody of a human mitochondrial outer membrane protein: cytochrome b5 type B (CYB5B), and an application of immunomagnetic beads combined with the CYB5B antibody (CYB5B magnetic bead) in extracting mitochondria from cells. Specifically, the CYB5B antibody purified by antigen affinity chromatography can specifically identify mitochondria in various organizations and organs, and almost does not take effect with other organelles. The purified CYB5B antibody can be combined with magnetic bead materials to obtain the CYB5B magnetic bead, and by using the CYB5B magnetic bead, complete mitochondria can be extracted from a few cells, cell number being more than or equal to 104. The antibody is particularly suitable for preparing mitochondria from cells cultured in laboratories, and the enrichment effect of the method is improved by 3 to 4 times of that of a traditional differential centrifugation method.

Description

technical field [0001] The present invention relates to the fields of biotechnology such as immunology and cytology, specifically: the present invention relates to the mitochondrial outer membrane protein cytochrome B5 isomer B (CYB5B), the preparation of CYB5B polyclonal antibody, and the formation of CYB5B antibody after coupling with magnetic materials Immunomagnetic beads (CYB5B magnetic beads) and a method for purifying mitochondria in cells using CYB5B magnetic beads. The mitochondria prepared by the method have a complete structure and high enrichment efficiency, and can be used in various fields of biotechnology. Background technique [0002] Mitochondria is an important organelle in eukaryotes, and its basic biological function is to synthesize the energy compound needed by cells, ATP. Mitochondria also have many important biological functions, including maintenance of ion homeostasis, substance metabolism and cell apoptosis. Therefore, mitochondrial dysfunction c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/06C07K17/00C07K1/22
Inventor 不公告发明人
Owner BEIJING PROTEIN INNOVATION
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