CN106928315B - Nucleic acid binding protein extraction method based on graphene oxide - Google Patents
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Abstract
A nucleic acid binding protein extraction method based on graphene oxide belongs to the technical field of novel protein enrichment. According to the invention, cells to be detected are added into cell lysis solution containing graphene oxide for lysis, and nucleic acid binding protein are adsorbed by graphene oxide to form a graphene oxide-nucleic acid binding protein compound. And (3) centrifuging to collect the compound, suspending the compound in protein denaturation liquid, carrying out ultrasonic treatment, and finally collecting the nucleic acid binding protein in the supernatant. The method has the advantages of low cost, simple operation and less than 1 minute in the extraction process, can simply and efficiently extract the nucleic acid binding protein, and has important significance for the research of the mass spectrum of the nucleic acid binding protein.
Description
Technical Field
The invention relates to a technology in the field of biological detection, in particular to a nucleic acid binding protein extraction method based on graphene oxide.
Background
Nucleic acid binding proteins, including DNA binding proteins and RNA binding proteins, are involved in regulating a variety of cellular functions, including DNA replication and transcription, RNA processing and translation, gene silencing, and maintenance of telomere length. RNA binding proteins can form ribonucleoprotein complexes with RNA to regulate RNA synthesis and degradation, and can also bind to non-coding RNA such as microRNA to regulate post-transcriptional processing of messenger RNA (mrna). DNA binding proteins, including nucleosome proteins and transcription factors, play a very important role in the process of DNA replication, transcription and repair. Loss of expression or function of nucleic acid binding proteins can lead to the development of a number of diseases, such as cancer and metabolic diseases. The important biological functions of nucleic acid binding proteins make the study of their intracellular protein profiles and their activation states under different physiopathological conditions particularly important.
Common methods for the study of nucleic acid binding proteins include chromatin immunoprecipitation, electrophoretic mobility assays, chromosome capture techniques, DNA or RNA affinity chromatography, and identification of nucleic acid binding proteins using mass spectrometry. These methods either study proteins bound to them, centered on a single or multiple genes, or study nucleic acid fragments bound to them, centered on a protein. These techniques are characterized by high specificity, but low throughput and are not useful as a means of nucleic acid binding proteomics research. In recent years, gene-centered methods have been developed to study DNA-binding proteins or RNA-binding proteins at high throughput. For example, protein microarray chip technology screens cell lysates for DNA-binding proteins by artificial synthesis and concatenation of hundreds of DNA fragments that have been shown to bind proteins (Hu et al, 2009). mRNA co-immunoprecipitation techniques utilize antibodies that can specifically bind mRNA, and RNA-binding proteins are identified by enriching for mRNA (Castello et al, 2012) (Baltz et al, 2012). Although a certain amount of nucleic acid binding protein can be identified by the two methods, the application of the two methods in nucleic acid binding proteomics research has great limitation due to complex operation and high cost. Until now, there is no general method for extracting nucleic acid binding protein, and therefore, it is necessary to develop a simple and efficient method for extracting nucleic acid binding protein.
Typically, nucleic acid binding proteins are in low abundance in cells, and thus enrichment for nucleic acid binding protein specificity will facilitate subsequent analysis thereof. Due to the nature of nucleic acid binding proteins that bind to nucleic acids, nucleic acid binding proteins can be extracted by enriching for nucleic acids. The traditional nucleic acid extraction method is based on a phenol-chloroform system, and most of nucleic acid binding protein is lost in the extraction process, so that a new method for enriching nucleic acid on the premise of ensuring that the nucleic acid binding protein is not lost needs to be developed.
Nanomaterials such as carbon nanotubes, graphene and graphene oxide are widely used in biomedical research, such as biosensors, drug carriers, etc., due to their excellent mechanical, thermal and electrical conductivity properties. Previous studies show that the nano material can stably adsorb single-stranded nucleic acid through acting force of pi-pi accumulation. In 2012, Zhang et al extracted nucleic acids from cells by using the property of carbon nanotubes to adsorb nucleic acids and identified 2595 proteins by mass spectrometry (Zhang et al, 2012), which is simple, but the specificity of nucleic acid binding protein extraction was only 24%.
Disclosure of Invention
The invention provides a graphene oxide-based nucleic acid binding protein extraction method aiming at the defect that nucleic acid binding protein cannot be extracted because only nucleic acid can be extracted and the nucleic acid binding protein is completely lost in the extraction process in the prior art, and the method is low in cost, simple to operate and less than 1 minute in the extraction process, can simply and efficiently extract the nucleic acid binding protein and is beneficial to research on the nucleic acid binding protein related to the occurrence and development of diseases.
The invention is realized by the following technical scheme:
the method comprises the steps of adding cells to be detected into cell lysis solution containing graphene oxide for lysis, centrifugally collecting and cracking the graphene oxide-nucleic acid binding protein complex obtained after lysis, suspending the graphene oxide-nucleic acid binding protein complex in protein denaturation solution, carrying out ultrasonic treatment, and collecting the nucleic acid binding protein in supernatant.
The cells to be detected are all types of animal cells, preferably are resuspended in buffer solution and slowly added with cell lysate, and more preferably are resuspended in PBS at 5x106Individual cells were resuspended in 20 μ L PBS.
The cell lysate specifically comprises: 5X10 per cleavage6For each cell, 500. mu.g of graphene oxide was dissolved in 500. mu.L of 250mM SDS.
The size of the graphene oxide is preferably less than 500 nm.
The centrifugal collection means that: slowly stirring by using a pipette tip to fully lyse cells, centrifugally collecting the graphene oxide-nucleic acid binding protein compound obtained after lysis, and washing the compound for 3 times by using ultrapure water.
The ultra-pure water washing is as follows: the complex was resuspended with ultrapure water in a 1.5mL EP tube and washed upside down without breaking up the complex.
The protein denaturation liquid is 6-8M urea, 10-100 mM Tris-HCl and pH 7.0-8.0.
The heavy suspension in the protein denaturation liquid specifically comprises the following steps: 300-500. mu.L of protein denaturant resuspend the complex in a 1.5mL EP tube.
The ultrasonic treatment is preferably carried out for 6-10 s under the environment of 200-400W.
The nucleic acid binding protein is identified by the following methods: the method of the invention is adopted to extract the nucleic acid binding protein of 293T cells, and the method of LC-MSMS is applied to identify the nucleic acid binding protein.
Technical effects
Compared with the prior art, the invention can quickly and effectively enrich nucleic acid in cells, including DNA and RNA, and extract nucleic acid binding protein by enriching nucleic acid. More specifically, the invention relates to the property that the nano material graphene oxide can adsorb nucleic acid, and the nucleic acid is enriched to nucleic acid binding protein from cells through enriching the nucleic acid. The method can be used for extracting nucleic acid binding protein of all animal cells, and is combined with quantitative proteomics technology to help the discovery and research of the nucleic acid binding protein related to the occurrence and development of diseases.
Drawings
FIG. 1 is a schematic flow diagram of the process;
FIG. 2 is a graphene oxide-nucleic acid binding protein complex;
in the figure: a is a picture of a graphene oxide-nucleic acid binding protein complex; b is the result of analyzing the compound by a confocal fluorescence microscope, wherein DIPA is a nucleic acid dye; c is the comparison of the extraction efficiency of RNA by using three nano materials of carboxylated graphene cG, oxidized graphene GO and a carboxylated carbon nanotube cCNT; wherein method A is the method used in the present invention, and method B is the method used in the literature; the efficiency of extracting RNA from graphene oxide is highest; d is the comparison of the DNA extraction efficiency of the three nano materials; the efficiency of extracting DNA from graphene oxide is highest; e is five nano materialsThe method comprises the following steps of comparing the RNA extraction efficiency of graphene G, carboxylated graphene cG, oxidized graphene GO and a highly carboxylated carbon nanotube hcCNT, wherein the RNA extraction efficiency of the oxidized graphene is the highest; f is prepared from graphene, graphene oxide and carbon nanotubes (5X 10)6The mass of nucleic acid binding protein extracted from the individual cells;
FIG. 3 is the result of SDS-PAGE combined with silver staining of proteins;
in the figure: a is a whole cell lysate; b is extracted nucleic acid binding protein; c is a negative control: protein extracted from the whole cell lysate after nuclease treatment;
FIG. 4 shows the identification of extracted nucleic acid binding proteins by LC-MSMS;
in the figure: a is the number of nucleic acid binding proteins identified for washing the complex for break-up and break-up-free methods, respectively; b is the content of the DNA binding protein histone H3 and the RNA binding protein PTBP1 in the whole cell lysate, the extracted nucleic acid binding protein and the cell supernatant after the nucleic acid binding protein is extracted respectively for detecting the protein immunoblotting.
Detailed Description
As shown in fig. 1, the present embodiment includes the following steps:
step 1, adding cells to be detected into a cell lysate containing graphene oxide for cracking:
1.1) preparing a cell lysate containing graphene oxide, wherein the component of the cell lysate is 250mM SDS, and a corresponding amount of graphene oxide is taken according to the number of cells to be lysed and dissolved in the cell lysate to make the final concentration of the graphene oxide 1mg/mL, generally 5x106500 μ g of graphene oxide was required for each cell.
1.2) resuspend the cells in 20. mu.L PBS and add them slowly to the cell lysate to lyse the cells, and lyse the cells thoroughly with gentle agitation with a pipette tip.
The cell to be detected is a 293T cell line.
1.3) centrifugation at 20000g for 5 minutes at 4 ℃ and discarding the supernatant.
1.4) collecting the graphene oxide-nucleic acid binding protein complex, as shown in FIG. 2A, and washing the complex 3 times upside down with ultrapure water without scattering the complex. As a result, as shown in fig. 2B, the nucleic acid was adsorbed by graphene oxide.
To date, there is still no general nucleic acid binding protein extraction method. Carbon nanotubes have been reported to be useful for the extraction of nucleic acid binding proteins (Zhang et al, 2012), and graphene oxide can adsorb more DNA and RNA than carbon nanotubes, as shown in fig. 2C-2E. The amount of nucleic acid binding protein extracted at the same number of cells was 4 times that of the carbon nanotubes, as shown in FIG. 2F. Thus, graphene oxide is more suitable for extraction of nucleic acid binding proteins.
Step 2, extracting nucleic acid binding protein:
2.1) the complex is washed and then resuspended in protein denaturation liquid, and the nucleic acid binding protein are desorbed from the graphene oxide by 400W ultrasonic for 10 s.
2.2) centrifuging at 4 ℃ and 20000g for 60 minutes, and collecting the supernatant;
2.3) the protein denaturant was replaced with ultrapure water using a 3K ultrafiltration tube and concentrated to a volume of 100. mu.L.
2.4) BCA assay for protein concentration, 2. mu.g of protein was taken for SDS-PAGE analysis. The results are shown in FIG. 3, which shows that the nucleic acid binding protein extracted by the present invention has high specificity.
Step 3, characterization of nucleic acid binding proteins:
3.1) extracting nucleic acid binding protein from 293T cells by the technology of the invention, and taking 30 mu g of protein for enzymolysis in solution.
3.2) adopting Q-active Orbitrap LC-MSMS to identify the extracted proteins, and identifying 2563 proteins in total.
The DNA binding protein histone H3, RNA binding protein PTBP1, was tested by Western blotting technique to be specifically enriched by the method developed by the present invention. The results are shown in FIG. 4, which demonstrates that the method of the present invention can specifically enrich nucleic acid binding proteins from cells with high efficiency.
The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (6)
1. A method for extracting nucleic acid binding protein based on graphene oxide is characterized in that cells to be detected are added into cell lysis solution containing graphene oxide which is smaller than 500nm and used for adsorbing nucleic acid for lysis, the graphene oxide-nucleic acid binding protein compound obtained after lysis is collected by centrifugation and is resuspended in protein denaturation solution and is subjected to ultrasonic treatment, and the nucleic acid binding protein in supernate is collected, so that the nucleic acid binding protein is obtained;
the cell lysate specifically comprises: 5X10 per cleavage6Cell lysis of 500 μ g graphene oxide in 500 μ L250 msds;
the centrifugal collection means that: slowly stirring by using a pipette tip to fully lyse cells, centrifugally collecting the graphene oxide-nucleic acid binding protein compound obtained after lysis, and washing the compound for 3 times by using ultrapure water.
2. The method of claim 1, wherein the test cells are all animal cells.
3. The method of claim 1 or 2, wherein the cell suspension of the test cells in PBS is 5x106Individual cells were resuspended in 20 μ L PBS.
4. The method for extracting nucleic acid binding protein according to claim 1, wherein the ultra-pure water washing is: the complex was resuspended with ultrapure water in a 1.5mL EP tube and washed upside down without breaking up the complex.
5. The method for extracting nucleic acid-binding protein according to claim 1, wherein the complex is resuspended in a protein denaturation solution containing 6 to 8M urea, 10 to 100mM Tris-HCl, and pH 7.0 to 8.0 and subjected to ultrasonic treatment.
6. The method for extracting nucleic acid binding protein according to claim 5, wherein the ultrasonic treatment conditions are as follows: 200-400W ultrasound for 6-10 s.
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Citations (3)
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| CN102978201A (en) * | 2012-12-24 | 2013-03-20 | 厦门大学 | Application of graphene in polymerase chain reaction as reinforcing agent |
| CN105713900A (en) * | 2016-03-29 | 2016-06-29 | 广州市玛达生物科技有限公司 | Nucleic acid extraction method based on magnetic graphene nano-composites |
| CN105950476A (en) * | 2016-06-08 | 2016-09-21 | 广东工业大学 | Method for brewer's yeast cell disruption based on collaborative driving of pulsed electric field and carbon nano tubes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978201A (en) * | 2012-12-24 | 2013-03-20 | 厦门大学 | Application of graphene in polymerase chain reaction as reinforcing agent |
| CN105713900A (en) * | 2016-03-29 | 2016-06-29 | 广州市玛达生物科技有限公司 | Nucleic acid extraction method based on magnetic graphene nano-composites |
| CN105950476A (en) * | 2016-06-08 | 2016-09-21 | 广东工业大学 | Method for brewer's yeast cell disruption based on collaborative driving of pulsed electric field and carbon nano tubes |
Non-Patent Citations (1)
| Title |
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| Highly Efficient Extraction of Cellular Nucleic Acid Associated Proteins in Vitro with Magnetic Oxidized Carbon Nanotubes;Yi Zhang等;《American Chemical Society》;20121112;第84卷(第23期);第10454左栏，图1 * |
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