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Preparation method of molecular biology bovine serum albumin

A technology of bovine serum albumin and molecular biology, which is applied in the field of preparation of molecular biological grade bovine serum albumin, can solve the problems of decreased detection sensitivity, delayed CT value, nuclease residue, etc., and achieves time-saving and labor-saving recovery efficiency and easy amplification Effects of scale and detection sensitivity improvement

Active Publication Date: 2018-11-23
珠海宝锐生物科技有限公司
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Problems solved by technology

[0007] At present, there are reports of diatomite (kaolin) or heparin combined with DEAE to remove nucleases alone, and no significant change has been detected by electrophoresis detection methods, but when the present invention uses fluorescent quantitative PCR method to detect nuclease residues, it is found that silicon There are still nuclease residues after the treatment of bovine serum albumin with algae earth or heparin, causing the CT value of the experiment to be delayed, the detection sensitivity to decrease, and the quantification to be inaccurate

Method used

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  • Preparation method of molecular biology bovine serum albumin
  • Preparation method of molecular biology bovine serum albumin
  • Preparation method of molecular biology bovine serum albumin

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1 uses diatomaceous earth to purify bovine serum albumin alone

[0045] When using diatomaceous earth to purify bovine serum albumin (remove nuclease in bovine serum albumin), the suspension used is Tris-HCl buffer solution, the concentration is 50mM, and pH is 7.8; the dialysate used is Tris-HCl buffer The solution has a concentration of 10 mM and a pH of 8.0; the diatomaceous earth and the suspension are sterilized at 121° C. for 30 minutes before use. The specific purification steps are:

[0046] (1) Mix 6g of diatomite with 60mL of suspension (the ratio of diatomite to suspension is 1:10w / v), place in a boiling water bath at 100°C for 5min, stir at room temperature for 5min, centrifuge at 5000rpm for 10min, discard Supernatant: After repeating the above washing steps 3 times, disperse the washed diatomaceous earth in 60mL suspension evenly to obtain the adsorbent dispersion, and store it at 4°C for later use;

[0047] (2) Dissolve and disperse 2.5g bovi...

Embodiment 2

[0053] Embodiment 2 uses Heparin-Sepharose chromatographic column to purify bovine serum albumin alone

[0054] When purifying bovine serum albumin (removing nuclease in bovine serum albumin) with Heparin-Sepharose chromatographic column, the chromatographic column balance liquid used is Tris-HCl buffer solution, and concentration is 20mM, and pH is 8.0; The solution is a Tris-HCl buffer solution with a concentration of 10 mM and a pH of 8.0; the balance solution is sterilized at 121°C for 30 minutes before use. The specific purification steps are:

[0055] (1) Disperse 2.5g bovine serum albumin evenly in 12.5mL equilibrium solution, filter it with a 0.45μm membrane, and use it as the loading sample;

[0056] (2) Equilibrate the Heparin-Sepharose chromatographic column 5 times CV with the balance liquid, and after the flow rate is 180mL / h, purify the loaded sample, collect the breakthrough peak, and elute with the balance liquid until the baseline is stable after loading the ...

Embodiment 3

[0060] Embodiment 3 uses the combination of diatomaceous earth and Heparin-Sepharose chromatographic column to purify bovine serum albumin

[0061] When using the combination of diatomaceous earth and Heparin-Sepharose chromatography column to purify bovine serum albumin (to remove nuclease in bovine serum albumin), the suspension used is Tris-HCl buffer solution with a concentration of 50mM and a pH of 7.8; The chromatography column balance solution used is Tris-HCl buffer solution, the concentration is 20mM, and the pH is 8.0; the dialysate used is Tris-HCl buffer solution, the concentration is 10mM, and the pH is 8.0; diatomaceous earth, suspension and balance solution Sterilize at 121°C for 30 minutes before use. The specific purification steps are:

[0062] (1) Mix 6g of diatomite with 60mL of suspension (the ratio of diatomite to suspension is 1:10w / v), place in a boiling water bath at 100°C for 5min, stir at room temperature for 5min, centrifuge at 5000rpm for 10min, d...

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Abstract

The invention belongs to the field of protein purification, and particularly relates to a preparation method of molecular biology bovine serum albumin. The preparation method of the molecular biologybovine serum albumin comprises the following steps of preparation of an adsorbent dispersion solution, purification of a bovine serum albumin sample by the adsorbent, treatment of the bovine serum albumin sample by a heparin-sepharose chromatographic column, ultrafiltration and concentration, dialysis and the like. The preparation method has the advantages that the bovine serum albumin sample is purified by combining diatomite and the heparin-sepharose chromatographic column, so that the nuclease in the bovine serum albumin sample can be removed, and the molecular biology bovine serum albuminis obtained; the whole purification process is performed under the low-temperature environment, and the activity of the bovine serum albumin is furthest maintained; the DEPC (diethylpyrocarbonate) treatment is not involved, and the pollution to environment is avoided; the trace nuclease in the bovine serum albumin sample can be removed, the time is shortened, the labor intensity is decreased, therecycling efficiency is high, and the scale is conveniently expanded.

Description

technical field [0001] The invention belongs to the field of protein purification, and in particular relates to a method for preparing molecular biological grade bovine serum albumin. Background technique [0002] Nuclease is an enzyme that can catalyze the hydrolysis of phosphodiester bonds in nucleotide chains (DNA and RNA), acting on the P-O position of phosphodiester bonds, and widely exists in animals, plants and in the air. Nucleases from different sources have different specificities and modes of action. Those that can only act on RNA are called ribonucleases (RNase), those that can only act on DNA are called deoxyribonucleases (DNase), and some nucleases can act on both RNA and DNA, so they are collectively called nucleases (nuclease). Nucleases can be divided into exonucleases and endonucleases according to the position of nuclease action. [0003] Since nucleases have a strong ability to degrade nucleic acids (DNA and RNA), false negative results are prone to oc...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K1/36C07K1/34C07K1/22C07K1/16
CPCC07K14/765
Inventor 时彦祎杨心意覃雨棠
Owner 珠海宝锐生物科技有限公司
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