阅读说明：本技术 一种柱式法从细胞中分离蛋白质的方法 (Method for separating protein from cells by column method ) 是由 卜汉蒙 于 2020-06-02 设计创作，主要内容包括：本发明公开了一种柱式法从细胞中分离蛋白质的方法,包括以下步骤：利用细胞裂解缓冲液对收集到的细胞进行裂解,使蛋白质、DNA、RNA、脂质和其他细胞组分溶于细胞裂解液中,得到粘性细胞裂解物,细胞裂解缓冲液包含表面活性剂和金属螯合剂；将粘性细胞裂解物转移至过滤装置内,再通过施加正向力或负向力,使粘性细胞裂解物通过滤装置,过滤装置内的多孔过滤介质会截留粘性细胞裂解物包含的基因组DNA和未裂解的细胞碎片,而蛋白质会穿过过滤装置后到达收集管,即为滤液；所述滤液即是从粘性细胞裂解物中分离的蛋白质提取物。本发明,能够提高蛋白质提取速度,增加蛋白质产量,提升实验体验度,以最小化提取时间最大化蛋白质产量。(The invention discloses a method for separating protein from cells by a column method, which comprises the following steps: lysing the collected cells with a cell lysis buffer, dissolving proteins, DNA, RNA, lipids and other cell components in the cell lysis buffer to obtain a viscous cell lysate, the cell lysis buffer comprising a surfactant and a metal chelator; transferring the viscous cell lysate into a filtering device, and applying positive force or negative force to enable the viscous cell lysate to pass through the filtering device, wherein a porous filtering medium in the filtering device can intercept genomic DNA and uncleaved cell fragments contained in the viscous cell lysate, and the protein can pass through the filtering device and then reach a collecting pipe, namely filtrate; the filtrate is the protein extract isolated from the viscous cell lysate. The method can improve the protein extraction speed, increase the protein yield, and improve the experimental experience, so as to minimize the extraction time and maximize the protein yield.)

1. A method for isolating a protein from a cell by a column method, comprising:

contacting cells with a cell lysis buffer, the cells lysing to produce a viscous cell lysate, wherein the cell lysis buffer comprises a surfactant and a metal chelator;

transferring the viscous cell lysate into a filtering device containing a porous filtering medium, and applying positive force or negative force to enable the viscous cell lysate to pass through the filtering device, wherein the porous filtering medium in the filtering device can retain genomic DNA and uncleaved cell fragments contained in the viscous cell lysate, and the protein can pass through the filtering device and then reach a collecting pipe, namely filtrate;

the filtrate, i.e. the protein extract isolated from the viscous cell lysate.

Wherein, the porous filter medium who uses among the filter equipment, the aperture is 20 ~ 60um, and thickness is 0.5mm ~ 20mm, sets up the bottom of filter equipment's inner chamber is used for holding back genome DNA and the cell debris that do not lyse in the viscous cell lysate.

2. The method of claim 1, wherein the cell lysis buffer does not contain a chaotropic agent.

3. The method of claim 1, wherein the cell lysis buffer comprises one or more surfactants.

4. The method of claim 2, wherein the surfactant comprises one or more of SDS, Triton X-100, NP-40, Tween-20, and digitonin.

5. The method of claim 1, wherein the filter media is made of a hydrophobic material or is made of a hydrophilic material.

6. The method of claim 1, wherein the cell lysis buffer is buffered at a pH of between 7.0 and 12.

7. The method of claim 1, wherein the cell is a vertebrate cell or a prokaryotic cell.

8. The method of claim 7, wherein the vertebrate cells are cultured cells or cells present in a tissue.

9. The method of claim 1, wherein the cell lysate contacts the cells in a volume of at least 20 ul.

10. The method of claim 1, wherein the positive force is applied by centrifugation for 30 seconds.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a method for separating protein from cells by a column method.

Background

Extraction of proteins from total cell lysates is one of the most commonly used methods in biomedical research. The first step is the release of the protein from the cell by chemical or physical means. Cell lysates containing denatured surfactants (e.g., SDS) and non-denatured surfactants (e.g., NP-40 and Tween-100) are often used in kits for commercial protein extraction. Although powerful surfactants (e.g., SDS) can effectively lyse cells, the presence of released genomic DNA in the cell lysate can make the lysate very viscous, difficult to pipette and may interfere with subsequent protein quantification.

One of the most widespread laboratory methods for separating proteins from total cell lysates is to lyse the cells using a solution containing a surfactant to extract the proteins and then reduce the viscosity of the solution using physical methods such as repeated sonication or repeated needle punching of the cell lysate through a fine injection needle to shear the DNA into smaller fragments, but such experimental methods are cumbersome and time consuming. Repeated sonication also generates a large amount of heat, which may inactivate the proteins.

In view of the above, there is an urgent need for improvement of the existing methods for separating proteins from cells, so as to increase the protein extraction speed, increase the protein yield, improve the experimental experience, minimize the extraction time, and maximize the protein yield.

Disclosure of Invention

The technical problems to be solved by the invention are that the existing method for separating protein from cells has long time for extracting protein, low yield and poor experimental experience.

In order to solve the technical problems, the technical scheme adopted by the invention is to provide a method for separating protein from cells by a column method, which comprises the following steps:

contacting cells with a cell lysis buffer, the cells lysing to produce a viscous cell lysate, wherein the cell lysis buffer comprises a surfactant and a metal chelator;

transferring the viscous cell lysate into a filtering device containing a porous filtering medium, and applying positive force or negative force to enable the viscous cell lysate to pass through the filtering device, wherein the porous filtering medium in the filtering device can retain genomic DNA and uncleaved cell fragments contained in the viscous cell lysate, and the protein can pass through the filtering device and then reach a collecting pipe, namely filtrate;

the filtrate, i.e. the protein extract isolated from the viscous cell lysate.

Wherein, the porous filter medium who uses among the filter equipment, the aperture is 20 ~ 60um, and thickness is 0.5mm ~ 20mm, sets up the bottom of filter equipment's inner chamber is used for holding back genome DNA and the cell debris that do not lyse in the viscous cell lysate.

In the above method, the cell lysis buffer does not contain a chaotropic agent.

In the above method, the cell lysis buffer contains one or more surfactants.

In the method, the surfactant comprises one or more of SDS, Triton X-100, NP-40, Tween-20 and digitonin.

In the above method, the filter medium is made of a hydrophobic material or a hydrophilic material.

In the above method, the cell lysis buffer is buffered at a pH of between 7.0 and 12.

In the above method, the cell is a vertebrate cell or a prokaryotic cell.

In the above method, the vertebrate cell may be a cultured cell or a cell present in a vertebrate tissue.

In the above method, the cell lysate is contacted with the cells in a volume of at least 20 ul.

In the method, the positive force is applied by centrifugation for 30 seconds.

Compared with the prior art, the invention firstly leads the cell to contact with the cell lysate for cracking, and leads the protein to be completely released to obtain the viscous cell lysate; and then transferring the viscous cell lysate into a filtering device, and separating and collecting the protein from the genomic DNA and the fragments in the viscous cell lysate by applying centrifugal force or vacuum suction, so that the extraction speed of the protein is increased, the protein yield is increased, the experimental experience is improved, the extraction time is minimized, and the protein yield is maximized.

Drawings

FIG. 1 is a schematic diagram showing the construction of a filtration apparatus for separating proteins from cells according to the present invention;

FIG. 2 is a flow chart of a method for separating proteins from cells by the column method of the present invention;

FIG. 3 is a Coomassie blue stained SDS-PAGE gel showing protein extraction from mouse cells using denatured cell lysis buffer of the present invention and conventional repeated sonication;

FIG. 4 is a Coomassie brilliant blue image of protein extraction from 293T cells using native cell lysis buffer run on SDS-PAGE gels in accordance with the present invention;

FIG. 5 is a Coomassie blue stained gel of SDS-PAGE extracted protein from 293T cells using cytoplasmic extraction buffer and nuclear extraction buffer according to the invention;

FIG. 6 is a Coomassie brilliant blue image of protein extracted from E.coli using bacterial cell lysis buffer stained on SDS-PAGE gel in accordance with the present invention;

FIG. 7 shows the results of the present invention and conventional sonication extraction of proteins by running on a 12% SDS-PAGE gel with Coomassie Brilliant blue staining. Proteins were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes and detected with anti-mouse MDM2, HDAC1 and ACTIN antibodies. Lane 1, NIH3T3 cell lysate prepared by repeated sonication; in lanes 2 and 3, proteins isolated using the denatured cell lysis buffer of the present invention;

FIG. 8 is a graph showing the results of WB proteins corresponding to those in FIG. 7;

FIG. 9 is a photograph of a 12% SDS-PAGE gel Coomassie Brilliant blue staining of proteins isolated using a commercially available kit. Lanes 1, 2 and 3 are commercial kit A; lanes 4, 5 and 6 are kit B.

Detailed Description

In the field of molecular biology at present, nucleic acid extraction is greatly changed, the traditional solution method is broken away, a silica gel membrane method is adopted, nucleic acid is separated by using high-salt and low-salt elution of the silica gel membrane, the method is commonly called a column membrane method, and a magnetic bead method which is convenient for mechanical automatic nucleic acid extraction appears later is also adopted. However, protein extraction has not been greatly developed in recent 30 years, and various solution methods have been used for protein isolation. Protein research is completed along with the completion of various genome sequencing, a large number of interesting genes are discovered, the functions of the genes are the key, and the subsequent functional research is functional executor protein, which is the reason of the current fire and heat of protein research. Protein research has to extract protein with high quality, but the traditional solution method for extracting protein needs repeated ultrasonic treatment, has complicated process and time consumption, can inactivate protein, and urgently needs to reform the existing protein extraction method.

The invention provides a method for separating protein from cells by a column method, which can greatly improve the protein extraction speed, the protein yield and the experimental experience, so as to minimize the extraction time and maximize the protein yield.

The method provided by the invention can rapidly extract protein from eukaryotic and prokaryotic cells, wherein the eukaryotic cells comprise vertebrate cells, such as murine cells (mice, rats and the like), human cells and any other mammalian cells, can be cultured cells, and can be cells existing in vertebrate tissues. Prokaryotic cells include unicellular bacteria and archaea, among others.

The invention is described in detail below with reference to the drawings and the detailed description.