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Preparation method of agglutinin-magnetosome composite
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A magnetosome, lectin technology, applied in separation methods, alkali metal compounds, chemical instruments and methods, etc.
Inactive Publication Date: 2017-05-31
SHAANXI EYOUNG TECH
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Problems solved by technology
[0003] However, the coupling of magnetosomes and lectins for the separation of glycoproteins has not been reported yet.
Method used
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Embodiment 1
[0016] A method for preparing a lectin-magnetosome complex, comprising the following steps:
[0017] (1) Suspend the bacteria in 1PBS buffer at a ratio of 1:10 (M:V), crush them for 3-4 times with a high-pressure homogenizer (pressure 120-140MPa), put them in a magnetic field, and magnetically separate them. Small body;
[0018] (2) Place the magnetosomes in 1×PBS buffer solution, ultrasonically wash 5-8 times, deionized water wash 3 times, vacuum freeze-dry, and store at -80°C for later use;
[0019] (3) Take 1 mg of magnetosomes and heat in 1 mL of 1% SDS solution at 100°C for 5 min;
[0021] (5) Magnetosomes were resuspended in 1mg / mLPE solution, 100W ultrasonic for 30min, and incubated in the dark for 2h, the magnetosomes were collected by the magnetic separator and washed 3 times with PB buffer before use;
[0022] (6) Add 1....
Embodiment 2
[0025] Utilize the preparation method of a kind of lectin-magnetosome complex as claimed in claim 1, it is characterized in that the separation of its protein comprises the following steps: take each 1mg of GA-magnetosome / WGA-PE-magnetosome, in 500 μL of 1% SDS solution was heated at 100°C for 5 minutes, and 5 μL of each sample was taken, 1 mg of OVA and ConA were dissolved in 1 mL of binding buffer [20 mmol / L Tris-HCl (pH 7.4), 0.15 mol / L NaCl, 1 mmol / LCaCl2, 1 mmol / LMgCl2, 1mmol / LMnCl2], wash the WGA-PE-magnetosome complex with binding buffer for 3 times, add 1mL of OVA and ConA protein mixture, react at 25℃, 100r / min for 1h, and the reaction is over Finally, unbound protein was collected, and the WGA-PE-magnetosome complex was washed 3 to 5 times with binding buffer, and 500 μL of elution buffer 20 mmol / LTris-HCl (pH7.4), 0.15 mol / L NaCl, 1 mmol was added / LCaCl2,
[0026] 1mmol / LMgCl2, 1mmol / LMnCl2, 100mmol / L N-acetylglucosamine, 25°C, 100r / min, react for 1h.
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Abstract
The invention relates to a preparation method of an agglutinin-magnetosome composite. The method comprises the following steps: suspending a strain in 1 PBS (phosphatebuffer solution) in an M:V ratio of 1:10, crushing with a high-pressure homogenizer (the pressure is 120-140 MPa) 3-4 times, putting into a magnetic field, and carrying out magnetic separation to obtain the magnetosome. Compared with the unmodified magnetosome, the modified PE (polyethylene)-magnetosome has better dispersity and can be coupled with more agglutinin. The prepared WGAPE-magnetosome can well separate the target glycoprotein, and avoids the influence of the magnetosome protein on the glycoprotein separation specificity.
Description
technical field [0001] The invention relates to a preparation method of a lectin-magnetosome complex. Background technique [0002] Lectin is a protein that can specifically bind to a certain type of glycosyl group. Covalently couplinglectin to a solid-phase carrier, using the affinity between lectin and sugar chains, can achieve the enrichment of specific sugar complexes. Collection and separation, which makes it widely used in research fields such as glycomics. Magnetosomes are magnetic nanoparticles produced by magnetotactic bacteria, which have naturally formed biofilms and have good biocompatibility. As an important part of biofilm, PE provides a large number of amino groups on the surface of magnetosomes, which can be used to couple functional molecules such as antibodies, enzymes, and drugs. In addition, magnetosomes also have the characteristics of uniform size, stable crystal form, and superparamagnetism, which arouse people's great enthusiasm for the study of ma...
Claims
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