CN112305215A - Kit for co-immunoprecipitation recovery and application thereof - Google Patents
Kit for co-immunoprecipitation recovery and application thereof Download PDFInfo
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Abstract
The invention is suitable for the technical field of biomedicine, and provides a kit for co-immunoprecipitation recovery and application thereof, wherein the kit comprises a cleaning solution, an eluent, a cell lysate, a cell crosslinking solution, immunomagnetic beads and a diluent; wherein the cell lysate comprises the following components: trihydroxymethyl aminomethane, sodium dodecyl sulfate, and ethylenediamine tetraacetic acid; the cell crosslinking solution is a paraformaldehyde solution; the immunomagnetic beads are protein A magnetic beads and/or protein G magnetic beads; the diluent comprises the following components: sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, ethylene diamine tetraacetic acid, tris (hydroxymethyl) aminomethane and sodium chloride. The kit provided by the invention has the characteristics of simple operation, short recovery period, high recovery efficiency and the like, and can effectively solve the problems of complex operation, long recovery period and high recovery background of the traditional co-immunoprecipitation recovery technology, thereby expanding the application of the co-immunoprecipitation in a gene expression regulation mechanism and related diseases.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a kit for co-immunoprecipitation recovery and application thereof.
Background
The principle of the co-immunoprecipitation technology is to fix a protein complex in a living cell state, then to precipitate the complex by an immunological method, to specifically enrich DNA, RNA and other proteins bound to a target protein, and to obtain information on the interaction of the protein with DNA, RNA and other proteins by purifying and detecting the target complex.
This technique analyzes the regulation of the activity of a target gene and the target gene of a known protein by the interaction of the protein with DNA, RNA and other proteins, and is widely used in studies on the regulation of in vivo transcriptional genes, the regulation of gene stability, the regulation of gene activity, and the like. This technique is often combined with DNA chip, gene sequencing and molecular cloning techniques.
DNA, RNA and proteins in eukaryotic cells are often present in the form of complexes. Therefore, the study of protein interactions with DNA, RNA and other proteins is a fundamental approach to elucidating the regulation mechanism of eukaryotic gene expression. Immunoprecipitation is currently the only method to study the interaction of proteins with DNA, RNA, and other proteins in vivo. The basic principle is to fix a protein complex in a living cell state, then precipitate the complex by an immunological method, specifically enrich DNA, RNA and other proteins combined with a target protein, and obtain information of the interaction of the protein with the DNA, RNA and other proteins by purifying and detecting the target molecule. Immunoprecipitation technology can not only detect the dynamic effects of proteins in vivo with DNA, RNA, and other proteins, but can also be used to study the relationship of various covalent modifications of histones to regulation of gene expression. With the further improvement of the immunoprecipitation technology, it will certainly play an increasingly important role in the gene expression regulation and control research.
Immunoprecipitation technology is a method developed based on in vivo analysis, and has become the main method for studying epigenetic and gene expression regulatory information in the past decade. This technique helps researchers judge what histone modifications will occur at a particular location in the genome in the nucleus. This technique helps researchers determine what proteins and RNAs are bound in the cell. This technique helps researchers determine what proteins are present in the cell to bind to other proteins. In recent years, the continuous development and improvement of this technology is a very effective tool for the in-depth analysis of major pathways for diseases such as cancer, cardiovascular diseases and central nervous system disorders.
However, because the immunoprecipitation recovery technique involves many steps and many reagents, many researchers cannot obtain an ideal recovery result by using the conventional immunoprecipitation recovery technique, and the existing immunoprecipitation recovery technique has the disadvantages of complicated operation, long recovery time, unstable recovery, and the like, so that improvement of the immunoprecipitation technique is an important requirement.
Disclosure of Invention
The embodiment of the invention aims to provide a kit for co-immunoprecipitation recovery, aiming at solving the problems in the background art.
The embodiment of the invention is realized in such a way that a kit for co-immunoprecipitation recovery comprises a cleaning solution, an eluent, a cell lysate, a cell crosslinking solution, immunomagnetic beads and a diluent;
the cell lysate comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L of trihydroxymethyl aminomethane, 0.1-10% of sodium dodecyl sulfate and 1-1000 mmol/L of ethylene diamine tetraacetic acid;
the cell crosslinking solution is a paraformaldehyde solution;
the immunomagnetic beads are protein A magnetic beads and/or protein G magnetic beads;
the diluent comprises the following components in molar concentration or mass percentage concentration: 0.01-1% of sodium dodecyl sulfate, 0.1-10% of polyethylene glycol octyl phenyl ether, 1-1000 mmol/L of ethylenediamine tetraacetic acid, 1-1000 mmol/L of tris (hydroxymethyl) aminomethane and 1-1000 mmol/L of sodium chloride.
Preferably, the cell lysate comprises the following components in molar concentration or mass percentage: 50-500 mmol/L of trihydroxymethyl aminomethane, 1-5% of sodium dodecyl sulfate and 10-100 mmol/L of ethylene diamine tetraacetic acid.
Preferably, the diluent comprises the following components in terms of molar concentration or mass percentage concentration: 0.01-0.05% of sodium dodecyl sulfate, 0.1-2% of polyethylene glycol octyl phenyl ether, 1-10 mmol/L of ethylenediamine tetraacetic acid, 10-100 mmol/L of tris (hydroxymethyl) aminomethane and 100-500 mmol/L of sodium chloride.
As a preferable scheme of the embodiment of the present invention, the cleaning solution includes:
cleaning fluid A; the cleaning solution A comprises the following components in molar concentration or mass percentage concentration: 0.01-1% of sodium dodecyl sulfate, 0.1-10% of polyethylene glycol octyl phenyl ether, 1-1000 mmol/L of ethylenediamine tetraacetic acid, 1-1000 mmol/L of tris (hydroxymethyl) aminomethane and 1-1000 mmol/L of sodium chloride;
cleaning fluid B; the cleaning liquid B comprises the following components in percentage by mol or mass: 0.01-1% of sodium dodecyl sulfate, 0.1-10% of polyethylene glycol octyl phenyl ether, 1-1000 mmol/L of ethylenediamine tetraacetic acid, 1-1000 mmol/L of tris (hydroxymethyl) aminomethane and 1-2000 mmol/L of sodium chloride; and
cleaning fluid C; the cleaning solution C comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L lithium chloride, 0.1-10% ethyl phenyl polyethylene glycol, 0.1-10% sodium deoxycholate, 1-1000 mmol/L ethylene diamine tetraacetic acid and 1-1000 mmol/L tris (hydroxymethyl) aminomethane.
Preferably, the cleaning solution a comprises the following components in terms of molar concentration or mass percentage concentration: 0.1-0.5% of sodium dodecyl sulfate, 1-5% of polyethylene glycol octyl phenyl ether, 1-10 mmol/L of ethylenediamine tetraacetic acid, 10-100 mmol/L of tris (hydroxymethyl) aminomethane and 100-500 mmol/L of sodium chloride.
Preferably, the cleaning liquid B comprises the following components in terms of molar concentration or mass percentage concentration: 0.1-0.5% of sodium dodecyl sulfate, 1-5% of polyethylene glycol octyl phenyl ether, 1-10 mmol/L of ethylenediamine tetraacetic acid, 10-100 mmol/L of tris (hydroxymethyl) aminomethane and 1000-2000 mmol/L of sodium chloride.
Preferably, the cleaning solution C comprises the following components in terms of molar concentration or mass percentage concentration: 100-500 mmol/L lithium chloride, 1-5% ethyl phenyl polyethylene glycol, 1-5% sodium deoxycholate, 1-10 mmol/L ethylene diamine tetraacetic acid and 10-100 mmol/L tris (hydroxymethyl) aminomethane.
As another preferable aspect of the embodiment of the present invention, the eluent includes one or more of a DNA eluent, an RNA eluent, and a protein eluent.
As another preferred scheme of the embodiment of the invention, the DNA eluent comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L of trihydroxymethyl aminomethane, 0.1-10% of sodium dodecyl sulfate, 1-1000 mmol/L of magnesium chloride and 1-1000 mmol/L of sodium chloride;
the RNA eluent comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L of trihydroxymethyl aminomethane, 0.1-10% of sodium dodecyl sulfate, 0.1-10% of ethyl phenyl polyethylene glycol, 1-1000 mmol/L of magnesium chloride and 1-1000 mmol/L of sodium chloride;
the protein eluent comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L of trihydroxymethyl aminomethane, 1-1000 mmol/L of sodium chloride, 0.1-10% of ethyl phenyl polyethylene glycol, 0.1-10% of sodium deoxycholate, 0.1-10% of sodium dodecyl sulfate and 1-1000 mmol/L of ethylene diamine tetraacetic acid.
Preferably, the DNA eluent comprises the following components in molar or mass percent concentrations: 10-100 mmol/L of trihydroxymethyl aminomethane, 1-5% of sodium dodecyl sulfate, 2-10 mmol/L of magnesium chloride and 50-500 mmol/L of sodium chloride.
Preferably, the RNA eluent comprises the following components in molar or mass percent concentrations: 10-100 mmol/L of trihydroxymethyl aminomethane, 2-5% of sodium dodecyl sulfate, 0.2-1% of ethyl phenyl polyethylene glycol, 2-10 mmol/L of magnesium chloride and 100-500 mmol/L of sodium chloride.
Preferably, the protein eluent comprises the following components in terms of molar concentration or mass percentage concentration: 50-100 mmol/L of trihydroxymethyl aminomethane, 100-500 mmol/L of sodium chloride, 1-5% of ethyl phenyl polyethylene glycol, 0.5-2% of sodium deoxycholate, 0.1-1% of sodium dodecyl sulfate and 40-400 mmol/L of ethylene diamine tetraacetic acid.
In another preferred embodiment of the present invention, the mass concentration of paraformaldehyde in the cell crosslinking liquid is 0.1-10%.
Preferably, the mass concentration of the paraformaldehyde in the cell crosslinking liquid is 0.5-5%.
Another object of the embodiments of the present invention is to provide an application of the above-mentioned kit in the interaction of protein and cellular molecules.
As another preferable mode of the embodiment of the present invention, the cellular molecule is any one of DNA, RNA, protein, and chromatin.
As another preferable solution of the embodiment of the present invention, the application includes the steps of:
mixing the immunomagnetic beads and the diluent, then placing the mixture on a magnetic rack for separation, and removing supernatant to obtain a first precipitate;
mixing the first precipitate with the diluent, and then incubating with an antibody to obtain an incubation liquid;
separating the incubation liquid on a magnetic frame, and removing the supernatant to obtain a second precipitate for later use;
after culturing the cells, fixing the cells by using the cell cross-linking solution, and adding a glycine solution to neutralize the cell cross-linking solution;
mixing the fixed cells with the cell lysate, and then carrying out ultrasonic and centrifugal treatment to obtain a sample;
diluting a sample by using the diluent to obtain a diluted sample;
mixing the second precipitate with the diluted sample, and separating in a magnetic rack to obtain a third precipitate;
washing the third precipitate with the cleaning solution;
mixing the washed third precipitate with the eluent, adding or not adding protease, placing the mixture on a magnetic frame for separation, and collecting supernatant;
purifying the cell molecules in the supernatant.
It is a further object of embodiments of the present invention to provide a use of the kit as described above for the preparation of a kit for the treatment and/or prevention of a disease or epigenetic test.
As another preferable mode of the embodiment of the present invention, the disease is any one of cancer, cardiovascular disease and central nervous system disorder.
According to the kit for co-immunoprecipitation recovery provided by the embodiment of the invention, by improving the formula of the reagents such as cell lysate, diluent and the like, the kit has the characteristics of simple operation, short recovery period, high recovery efficiency and the like, and can effectively solve the problems of complex operation, long recovery period and high recovery background of the traditional co-immunoprecipitation recovery technology, so that the application of the kit in a gene expression regulation mechanism and related diseases can be enlarged.
Drawings
FIG. 1 is a graph showing the results of comparing the recovery cycle of the protein-related DNA recovery method provided in example 6 with that of the conventional co-immunoprecipitation recovery technique.
FIG. 2 is a graph showing the recovery results of the method for recovering a protein-related DNA provided in example 6.
FIG. 3 is a graph showing the recovery results of protein-related DNA by conventional co-immunoprecipitation recovery techniques.
FIG. 4 is a graph comparing the recovery cycles of the protein-related RNA recovery method provided in example 7 with those of the conventional co-immunoprecipitation recovery technique.
FIG. 5 is a graph showing the recovery results of the method for recovering protein-related RNA provided in example 7.
FIG. 6 is a graph showing the recovery results of protein-associated RNA from conventional co-immunoprecipitation recovery techniques.
FIG. 7 is a graph showing the results of comparing the recovery cycle of the protein-related protein recovery method provided in example 8 with that of the conventional co-immunoprecipitation recovery technique.
FIG. 8 is a graph showing the results of the recovery of the protein-related protein provided in example 8.
FIG. 9 is a graph showing the recovery results of protein-related proteins by a conventional co-immunoprecipitation recovery technique.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The embodiment provides a kit for co-immunoprecipitation recovery, which comprises a cleaning solution, an eluent, a cell lysate, a cell crosslinking solution, immunomagnetic beads and a diluent; the cleaning liquid comprises a cleaning liquid A, a cleaning liquid B and a cleaning liquid C; the eluent comprises a DNA eluent, an RNA eluent and a protein eluent, and the eluent can only comprise one or two of the DNA eluent, the RNA eluent and the protein eluent and can be set according to actual requirements; the immunomagnetic beads are protein A magnetic beads.
The cell lysis solution is prepared from trihydroxymethyl aminomethane, sodium dodecyl sulfate, ethylene diamine tetraacetic acid and water, and the pH of the cell lysis solution can be adjusted to 7 by acid or alkali. Wherein the molar concentration of the trihydroxymethyl aminomethane in the cell lysis solution is 1mmol/L, the mass percentage concentration of the lauryl sodium sulfate is 10 percent, and the molar concentration of the ethylene diamine tetraacetic acid is 1 mmol/L.
The cell crosslinking solution is a paraformaldehyde solution, and the mass concentration of the cell crosslinking solution is 0.1%.
The diluent is prepared from sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, tris (hydroxymethyl) aminomethane, sodium chloride and water, and the pH of the diluent can be adjusted to 7 by acid or alkali. Wherein the mass percent concentration of the lauryl sodium sulfate in the diluent is 0.01 percent, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 0.1 percent, the molar concentration of the ethylene diamine tetraacetic acid is 1000mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 1000mmol/L, and the molar concentration of the sodium chloride is 1000 mmol/L.
The cleaning solution A is prepared from sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, ethylene diamine tetraacetic acid, tris (hydroxymethyl) aminomethane, sodium chloride and water, and the pH of the cleaning solution A can be adjusted to 7 by acid or alkali. Wherein, the mass percent concentration of the lauryl sodium sulfate in the cleaning solution A is 0.01 percent, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 0.1 percent, the molar concentration of the ethylene diamine tetraacetic acid is 1000mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 1000mmol/L, and the molar concentration of the sodium chloride is 1000 mmol/L.
The cleaning solution B is prepared from sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, ethylene diamine tetraacetic acid, tris (hydroxymethyl) aminomethane, sodium chloride and water, and the pH of the cleaning solution B can be adjusted to 7 by acid or alkali. Wherein the mass percent concentration of the lauryl sodium sulfate in the cleaning solution B is 0.01 percent, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 0.1 percent, the molar concentration of the ethylene diamine tetraacetic acid is 1mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 1000mmol/L, and the molar concentration of the sodium chloride is 2000 mmol/L.
The cleaning solution C is prepared from lithium chloride, ethyl phenyl polyethylene glycol, sodium deoxycholate, ethylene diamine tetraacetic acid, tris (hydroxymethyl) aminomethane and water, and the pH of the cleaning solution C can be adjusted to 7 by using acid or alkali. Wherein the molar concentration of lithium chloride in the cleaning solution C is 1mmol/L, the mass percent concentration of ethyl phenyl polyethylene glycol is 0.1%, the mass percent concentration of sodium deoxycholate is 0.1%, the molar concentration of ethylene diamine tetraacetic acid is 1000mmol/L, and the molar concentration of tris (hydroxymethyl) aminomethane is 1000 mmol/L.
The DNA eluent is prepared from tris, sodium dodecyl sulfate, magnesium chloride, sodium chloride and water, and the pH of the DNA eluent can be adjusted to 7 with an acid or an alkali. Wherein the molar concentration of the trihydroxymethyl aminomethane in the DNA eluent is 1mmol/L, the mass percent concentration of the sodium dodecyl sulfate is 10 percent, the molar concentration of the magnesium chloride is 1mmol/L, and the molar concentration of the sodium chloride is 1 mmol/L.
The RNA eluent is prepared from trihydroxymethyl aminomethane, sodium dodecyl sulfate, ethylphenyl polyethylene glycol, magnesium chloride, sodium chloride and water, and the pH of the RNA eluent can be adjusted to 7 by acid or alkali. Wherein, the molar concentration of the trihydroxymethyl aminomethane in the RNA eluent is 1mmol/L, the mass percent concentration of the lauryl sodium sulfate is 10%, the mass percent concentration of the ethyl phenyl polyethylene glycol is 10%, the molar concentration of the magnesium chloride is 1mmol/L, and the molar concentration of the sodium chloride is 1 mmol/L.
The protein eluent is prepared from trihydroxymethyl aminomethane, sodium chloride, ethyl phenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, ethylene diamine tetraacetic acid and water, and can be adjusted to pH 7 with acid or alkali. Wherein the molar concentration of the trihydroxymethyl aminomethane in the protein eluent is 1mmol/L, the molar concentration of the sodium chloride is 1mmol/L, the mass percent concentration of the ethyl phenyl polyethylene glycol is 10%, the mass percent concentration of the sodium deoxycholate is 10%, the mass percent concentration of the sodium dodecyl sulfate is 10%, and the molar concentration of the ethylene diamine tetraacetic acid is 1000 mmol/L.
Example 2
The embodiment provides a kit for co-immunoprecipitation recovery, which comprises a cleaning solution, an eluent, a cell lysate, a cell crosslinking solution, immunomagnetic beads and a diluent; the cleaning liquid comprises a cleaning liquid A, a cleaning liquid B and a cleaning liquid C; the eluent comprises DNA eluent, RNA eluent and protein eluent; the immunomagnetic beads are protein G magnetic beads. The preparation method of each reagent is the same as that of example 1, and the differences are as follows:
the pH of the cell lysate is 9, wherein the molar concentration of the trihydroxymethyl aminomethane is 1000mmol/L, the mass percentage concentration of the lauryl sodium sulfate is 0.1%, and the molar concentration of the ethylene diamine tetraacetic acid is 1000 mmol/L.
The cell crosslinking solution is paraformaldehyde solution, and the mass concentration of the cell crosslinking solution is 10%.
The pH of the diluted solution is 9, wherein the mass percent concentration of the lauryl sodium sulfate is 1%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 10%, the molar concentration of the ethylene diamine tetraacetic acid is 1mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 1mmol/L, and the molar concentration of the sodium chloride is 1 mmol/L.
The pH of the cleaning solution A is 9, wherein the mass percent concentration of the lauryl sodium sulfate is 1%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 10%, the molar concentration of the ethylene diamine tetraacetic acid is 1mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 1mmol/L, and the molar concentration of the sodium chloride is 1 mmol/L.
The pH of the cleaning solution B is 9, wherein the mass percent concentration of the lauryl sodium sulfate is 1%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 10%, the molar concentration of the ethylene diamine tetraacetic acid is 1000mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 1mmol/L, and the molar concentration of the sodium chloride is 1 mmol/L.
The pH of the cleaning solution C is 9, wherein the molar concentration of lithium chloride is 1000mmol/L, the mass percent concentration of ethyl phenyl polyethylene glycol is 10%, the mass percent concentration of sodium deoxycholate is 10%, the molar concentration of ethylene diamine tetraacetic acid is 1mmol/L, and the molar concentration of tris (hydroxymethyl) aminomethane is 1 mmol/L.
The DNA eluate had a pH of 9, wherein the molar concentration of tris (hydroxymethyl) aminomethane was 1000mmol/L, the mass percentage concentration of sodium dodecyl sulfate was 0.1%, the molar concentration of magnesium chloride was 1000mmol/L, and the molar concentration of sodium chloride was 1000 mmol/L.
The pH of the RNA eluent is 9, wherein the molar concentration of the trihydroxymethyl aminomethane is 1000mmol/L, the mass percent concentration of the sodium dodecyl sulfate is 0.1%, the mass percent concentration of the ethyl phenyl polyethylene glycol is 0.1%, the molar concentration of the magnesium chloride is 1000mmol/L, and the molar concentration of the sodium chloride is 1000 mmol/L.
The pH of the protein eluent is 9, wherein the molar concentration of the trihydroxymethyl aminomethane is 1000mmol/L, the molar concentration of the sodium chloride is 1000mmol/L, the mass percent concentration of 0.1% of ethyl phenyl polyethylene glycol is 0.1%, the mass percent concentration of sodium deoxycholate is 0.1%, the mass percent concentration of the sodium dodecyl sulfate is 0.1%, and the molar concentration of the ethylene diamine tetraacetic acid is 1000 mmol/L.
Example 3
The embodiment provides a kit for co-immunoprecipitation recovery, which comprises a cleaning solution, an eluent, a cell lysate, a cell crosslinking solution, immunomagnetic beads and a diluent; the cleaning liquid comprises a cleaning liquid A, a cleaning liquid B and a cleaning liquid C; the eluent comprises DNA eluent, RNA eluent and protein eluent; the immunomagnetic beads are a mixture of protein A magnetic beads and protein G magnetic beads. The preparation method of each reagent is the same as that of example 1, and the differences are as follows:
the pH of the cell lysate is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 50mmol/L, the mass percentage concentration of the sodium dodecyl sulfate is 1%, and the molar concentration of the ethylene diamine tetraacetic acid is 10 mmol/L.
The cell crosslinking solution is a paraformaldehyde solution, and the mass concentration of the cell crosslinking solution is 0.5%.
The pH of the diluted solution is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.01%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 0.1%, the molar concentration of the ethylene diamine tetraacetic acid is 1, the molar concentration of the tris (hydroxymethyl) aminomethane is 10mmol/L, and the molar concentration of the sodium chloride is 100 mmol/L.
The pH of the cleaning solution A is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.1%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 1%, the molar concentration of the ethylene diamine tetraacetic acid is 1mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 10mmol/L, and the molar concentration of the sodium chloride is 100 mmol/L.
The pH of the cleaning solution B is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.1%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 1%, the molar concentration of the ethylene diamine tetraacetic acid is 1mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 10mmol/L, and the molar concentration of the sodium chloride is 1000 mmol/L.
The pH of the cleaning solution C is 8, wherein the molar concentration of lithium chloride is 100mmol/L, the mass percent concentration of ethyl phenyl polyethylene glycol is 1%, the mass percent concentration of sodium deoxycholate is 1%, the molar concentration of ethylene diamine tetraacetic acid is 1mmol/L, and the molar concentration of tris (hydroxymethyl) aminomethane is 10 mmol/L.
The DNA eluate has a pH of 8, wherein the molar concentration of tris (hydroxymethyl) aminomethane is 10mmol/L, the mass percentage concentration of sodium dodecyl sulfate is 1%, the molar concentration of magnesium chloride is 2mmol/L, and the molar concentration of sodium chloride is 50 mmol/L.
The pH of the RNA eluent is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 10mmol/L, the mass percent concentration of the sodium dodecyl sulfate is 2%, the mass percent concentration of the ethyl phenyl polyethylene glycol is 0.2%, the molar concentration of the magnesium chloride is 2mmol/L, and the molar concentration of the sodium chloride is 100 mmol/L.
The pH of the protein eluent is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 50mmol/L, the molar concentration of the sodium chloride is 100mmol/L, the mass percent concentration of the ethyl phenyl polyethylene glycol is 1%, the mass percent concentration of the sodium deoxycholate is 0.2%, the mass percent concentration of the sodium dodecyl sulfate is 0.1%, and the molar concentration of the ethylene diamine tetraacetic acid is 40 mmol/L.
Example 4
The embodiment provides a kit for co-immunoprecipitation recovery, which comprises a cleaning solution, an eluent, a cell lysate, a cell crosslinking solution, immunomagnetic beads and a diluent; the cleaning liquid comprises a cleaning liquid A, a cleaning liquid B and a cleaning liquid C; the eluent comprises DNA eluent, RNA eluent and protein eluent; the immunomagnetic beads are a mixture of protein A magnetic beads and protein G magnetic beads. The preparation method of each reagent is the same as that of example 1, and the differences are as follows:
the pH of the cell lysate is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 500mmol/L, the mass percentage concentration of the sodium dodecyl sulfate is 5%, and the molar concentration of the ethylene diamine tetraacetic acid is 100 mmol/L.
The cell crosslinking solution is a paraformaldehyde solution, and the mass concentration of the cell crosslinking solution is 5%.
The pH of the diluted solution is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.05 percent, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 2 percent, the molar concentration of the ethylene diamine tetraacetic acid is 10mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 100mmol/L, and the molar concentration of the sodium chloride is 500 mmol/L.
The pH of the cleaning solution A is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.5%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 5%, the molar concentration of the ethylene diamine tetraacetic acid is 10mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 100mmol/L, and the molar concentration of the sodium chloride is 500 mmol/L.
The pH of the cleaning solution B is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.5%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 5%, the molar concentration of the ethylene diamine tetraacetic acid is 10mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 100mmol/L, and the molar concentration of the sodium chloride is 2000 mmol/L.
The pH of the cleaning solution C is 8, wherein the molar concentration of lithium chloride is 500mmol/L, the mass percent concentration of ethyl phenyl polyethylene glycol is 5%, the mass percent concentration of sodium deoxycholate is 5%, the molar concentration of ethylene diamine tetraacetic acid is 10mmol/L, and the molar concentration of tris (hydroxymethyl) aminomethane is 100 mmol/L.
The DNA eluate had a pH of 8, wherein the molar concentration of tris (hydroxymethyl) aminomethane was 100mmol/L, the mass percentage concentration of sodium dodecyl sulfate was 5%, the molar concentration of magnesium chloride was 100mmol/L, and the molar concentration of sodium chloride was 500 mmol/L.
The pH of the RNA eluent is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 100mmol/L, the mass percent concentration of the sodium dodecyl sulfate is 5%, the mass percent concentration of the ethyl phenyl polyethylene glycol is 1%, the molar concentration of the magnesium chloride is 10mmol/L, and the molar concentration of the sodium chloride is 500 mmol/L.
The pH of the protein eluent is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 100mmol/L, the molar concentration of the sodium chloride is 800mmol/L, the mass percent concentration of 5% of ethyl phenyl polyethylene glycol is 2% of sodium deoxycholate is 1%, the mass percent concentration of the sodium dodecyl sulfate is 1%, and the molar concentration of the ethylene diamine tetraacetic acid is 400 mmol/L.
Example 5
The embodiment provides a kit for co-immunoprecipitation recovery, which comprises a cleaning solution, an eluent, a cell lysate, a cell crosslinking solution, immunomagnetic beads and a diluent; the cleaning liquid comprises a cleaning liquid A, a cleaning liquid B and a cleaning liquid C; the eluent comprises DNA eluent, RNA eluent and protein eluent; the immunomagnetic beads are a mixture of protein A magnetic beads and protein G magnetic beads. The preparation method of each reagent is the same as that of example 1, and the differences are as follows:
the pH of the cell lysate is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 250mmol/L, the mass percentage concentration of the sodium dodecyl sulfate is 3%, and the molar concentration of the ethylene diamine tetraacetic acid is 50 mmol/L.
The cell crosslinking solution is a paraformaldehyde solution, and the mass concentration of the cell crosslinking solution is 3%.
The pH of the diluted solution is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.03%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 1%, the molar concentration of the ethylene diamine tetraacetic acid is 5mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 50mmol/L, and the molar concentration of the sodium chloride is 300 mmol/L.
The pH of the cleaning solution A is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.3%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 3%, the molar concentration of the ethylene diamine tetraacetic acid is 5mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 50mmol/L, and the molar concentration of the sodium chloride is 300 mmol/L.
The pH value of the cleaning liquid B is 8, wherein the mass percent concentration of the lauryl sodium sulfate is 0.3%, the mass percent concentration of the polyethylene glycol octyl phenyl ether is 3%, the molar concentration of the ethylene diamine tetraacetic acid is 5mmol/L, the molar concentration of the tris (hydroxymethyl) aminomethane is 50mmol/L, and the molar concentration of the sodium chloride is 1500 mmol/L.
The pH of the cleaning solution C is 8, wherein the molar concentration of lithium chloride is 300mmol/L, the mass percent concentration of ethyl phenyl polyethylene glycol is 3%, the mass percent concentration of sodium deoxycholate is 3%, the molar concentration of ethylene diamine tetraacetic acid is 5mmol/L, and the molar concentration of tris (hydroxymethyl) aminomethane is 50 mmol/L.
The DNA eluent has pH of 8, wherein the molar concentration of trihydroxymethyl aminomethane is 50mmol/L, the mass percent concentration of sodium dodecyl sulfate is 3%, the molar concentration of magnesium chloride is 50mmol/L, and the molar concentration of sodium chloride is 250 mmol/L.
The pH of the RNA eluent is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 50mmol/L, the mass percent concentration of the sodium dodecyl sulfate is 3%, the mass percent concentration of the ethyl phenyl polyethylene glycol is 0.6%, the molar concentration of the magnesium chloride is 6mmol/L, and the molar concentration of the sodium chloride is 300 mmol/L.
The pH of the protein eluent is 8, wherein the molar concentration of the trihydroxymethyl aminomethane is 80mmol/L, the molar concentration of the sodium chloride is 450mmol/L, the mass percent concentration of the ethyl phenyl polyethylene glycol is 3 percent, the mass percent concentration of the sodium deoxycholate is 1 percent, the mass percent concentration of the sodium dodecyl sulfate is 0.5 percent, and the molar concentration of the ethylene diamine tetraacetic acid is 200 mmol/L.
Example 6
This example provides the use of the kit of example 5 above in protein-DNA interaction, wherein the method for recovering protein-associated DNA comprises the steps of:
s1, mixing the immunomagnetic beads uniformly, adding a proper amount of the immunomagnetic beads into the diluent, slightly blowing and beating for several times, placing the mixture on a magnetic rack for separation for 1min, and removing the supernatant to obtain a first precipitate.
And S2, mixing the first precipitate with the diluent, and then incubating the mixture with an anti-X gene antibody at 4 ℃ for 1h to obtain an incubation solution. In addition, IgG was added as a negative control in the same manner as in this step.
S3, placing the incubation liquid on a magnetic rack for separation for 1min, and removing the supernatant to obtain a second precipitate for later use.
S4, culturing the cells, fixing the cells by the cell cross-linking solution, and adding glycine solution to neutralize the cell cross-linking solution.
S5, washing the fixed cells with precooled PBS for 3 times, then adding a proper amount of precooled PBS, scraping the cells with a cell scraper, and collecting the cells into a centrifuge tube; then, the cell lysate is added into a centrifuge tube to be mixed with cells, and the mixture is placed on an ice bath to be incubated for 10 minutes so as to fully lyse the cells, then the cells are processed by ultrasonic treatment and 13000g centrifugation for 5 minutes, and the supernatant is taken out to a 2mL centrifuge tube to obtain a sample.
And S6, diluting the sample by using the diluent to obtain a diluted sample. And taking part of the diluted sample as an Input sample for subsequent detection.
S7, mixing the second precipitate and the diluted sample at 4 deg.C for 3 hr or overnight, separating for 1min with a magnetic frame, and removing the liquid carefully without touching the magnetic beads to obtain a third precipitate.
S8, washing the third precipitate 1 time with the washing solution a, the washing solution B, and the washing solution C in this order.
S9, properly bathing and melting the DNA eluent, mixing the DNA eluent with the washed third precipitate, adding proteinase K, uniformly mixing, incubating at 65 ℃ for 1 hour, then placing the mixture on a magnetic frame for separation for 1min, and collecting the supernatant; proteinase K was also added to the Input samples and incubated at 65 ℃ for 1 hour.
S10, purifying the DNA in the supernatant by a phenol chloroform method to obtain DNA precipitate; then, the DNA pellet was resuspended in a small amount of water, and detection primers were designed for quantitative PCR detection.
Example 7
This example provides the use of the kit of example 5 above for protein-RNA interaction, wherein the method for recovering protein-associated RNA comprises the steps of:
s1, mixing the immunomagnetic beads uniformly, adding a proper amount of the immunomagnetic beads into the diluent, slightly blowing and beating for several times, placing the mixture on a magnetic rack for separation for 1min, and removing the supernatant to obtain a first precipitate.
And S2, mixing the first precipitate with the diluent, and then incubating the mixture with an anti-X gene antibody at 4 ℃ for 1h to obtain an incubation solution. In addition, IgG was added as a negative control in the same manner as in this step.
S3, placing the incubation liquid on a magnetic rack for separation for 1min, and removing the supernatant to obtain a second precipitate for later use.
S4, culturing the cells, fixing the cells by the cell cross-linking solution, and adding glycine solution to neutralize the cell cross-linking solution.
S5, washing the fixed cells with precooled PBS for 3 times, then adding a proper amount of precooled PBS, scraping the cells with a cell scraper, and collecting the cells into a centrifuge tube; then, the cell lysate is added into a centrifuge tube to be mixed with cells, and the mixture is placed on an ice bath to be incubated for 10 minutes so as to fully lyse the cells, then the cells are processed by ultrasonic treatment and centrifugation at 12000g for 5 minutes, and the supernatant is taken out to be placed into a 2mL centrifuge tube to obtain a sample.
And S6, diluting the sample by using the diluent to obtain a diluted sample. And taking part of the diluted sample as an Input sample for subsequent detection.
S7, mixing the second precipitate and the diluted sample at 4 deg.C for 3 hr or overnight, separating for 1min with a magnetic frame, and removing the liquid carefully without touching the magnetic beads to obtain a third precipitate.
S8, washing the third precipitate 1 time with the washing solution a, the washing solution B, and the washing solution C in this order.
S9, properly bathing and melting the RNA eluent, mixing the RNA eluent with the washed third precipitate, adding proteinase K, uniformly mixing, incubating at 65 ℃ for 1 hour, then placing the mixture on a magnetic frame for separation for 1min, and collecting the supernatant; proteinase K was also added to the Input samples and incubated at 65 ℃ for 1 hour.
S10, purifying the RNA in the supernatant by using a Trizol method to obtain an RNA precipitate; then, the RNA pellet was resuspended in a small amount of RNase-free water, and detection primers were designed for quantitative PCR detection.
Example 8
This example provides the use of the kit of example 5 above for the interaction of proteins with other proteins, wherein the method for recovering protein-related proteins comprises the steps of:
s1, mixing the immunomagnetic beads uniformly, adding a proper amount of the immunomagnetic beads into the diluent, slightly blowing and beating for several times, placing the mixture on a magnetic rack for separation for 1min, and removing the supernatant to obtain a first precipitate.
And S2, mixing the first precipitate with the diluent, and then incubating the mixture with an anti-X gene antibody at 4 ℃ for 1h to obtain an incubation solution. In addition, IgG was added as a negative control in the same manner as in this step.
S3, placing the incubation liquid on a magnetic rack for separation for 1min, and removing the supernatant to obtain a second precipitate for later use.
S4, culturing the cells, fixing the cells by the cell cross-linking solution, and adding glycine solution to neutralize the cell cross-linking solution.
S5, washing the fixed cells with precooled PBS for 3 times, then adding a proper amount of precooled PBS, scraping the cells with a cell scraper, and collecting the cells into a centrifuge tube; then, the cell lysate is added into a centrifuge tube to be mixed with cells, and the mixture is placed on an ice bath to be incubated for 10 minutes so as to fully lyse the cells, then the cells are processed by ultrasonic treatment and 14000g centrifugation for 5 minutes, and the supernatant is taken out to be placed into a 2mL centrifuge tube to obtain a sample.
And S6, diluting the sample by using the diluent to obtain a diluted sample. And taking part of the diluted sample as an Input sample for subsequent detection.
S7, mixing the second precipitate and the diluted sample at 4 deg.C for 3 hr or overnight, separating for 1min with a magnetic frame, and removing the liquid carefully without touching the magnetic beads to obtain a third precipitate.
S8, washing the third precipitate 1 time with the washing solution a, the washing solution B, and the washing solution C in this order.
S9, properly bathing and melting the protein eluent, mixing the protein eluent with the washed third precipitate, placing the mixture into boiling water for treatment for 5min, then placing the mixture into a magnetic frame for separation for 1min, and collecting the supernatant.
S10, purifying, recovering and detecting the protein in the supernatant.
Experimental example:
first, the recovery cycle of the method for recovering a protein-related DNA provided in example 6 was compared with that of the conventional co-immunoprecipitation recovery technique, and the results are shown in FIG. 1, in which the recovery time of the method for recovering a protein-related DNA provided in example 6 was shortened from 17 hours to 8 hours, thereby completing the recovery. In addition, the recovery efficiency and the recovery background of the recovery method of protein-related DNA provided in example 6 are shown in FIG. 2, and the recovery efficiency and the recovery background of the conventional co-immunoprecipitation recovery technique are shown in FIG. 3. In the figure, X antibody is X gene antibody group; IgG, control antibody group; e gene: x gene-related gene DNA; f gene-unrelated gene DNA. As can be seen from FIGS. 1 to 3, the kit and the method for recovering protein-related DNA provided by the embodiment of the invention have the advantages of short recovery period, high recovery efficiency and low recovery background.
Second, comparing the recovery cycle of the protein-related RNA recovery method provided in example 7 with that of the conventional co-immunoprecipitation recovery technique, the results are shown in fig. 4, and the recovery of the protein-related RNA provided in example 7 was completed by shortening the original 17 hours to 8 hours. In addition, the recovery efficiency and the recovery background of the protein-related RNA recovery method provided in example 7 are shown in FIG. 5, and the recovery efficiency and the recovery background of the conventional co-immunoprecipitation recovery technique are shown in FIG. 6. In the figure, X antibody is X gene antibody group; IgG, control antibody group; e gene: x gene-related gene RNA; f gene-RNA of an unrelated gene. As can be seen from FIGS. 4 to 6, the kit and the method for recovering protein-related RNA provided by the embodiment of the invention have the advantages of short recovery period, high recovery efficiency and low recovery background.
Third, comparing the recovery cycle of the method for recovering a protein-related protein provided in example 8 with that of the conventional co-immunoprecipitation recovery technique, the results are shown in fig. 7, and the recovery of the protein-related protein provided in example 8 was completed by shortening the original 16 hours to 7 hours. In addition, the recovery results of the recovery method of protein-related proteins provided in example 8 are shown in FIG. 8, and the recovery results of the conventional co-immunoprecipitation recovery technique are shown in FIG. 9. In the figure, Anti-Z is a Z gene antibody group; IgG, control antibody group, Input, background group; gene Z: a Z gene protein; GAPDH: unrelated control proteins. As can be seen from FIGS. 7 to 9, the kit and the method for recovering protein-related protein provided by the embodiment of the invention have the advantages of short recovery period and high recovery efficiency.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A kit for co-immunoprecipitation recovery, comprising a washing solution and an eluent, characterized by further comprising:
a cell lysate; the cell lysate comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L of trihydroxymethyl aminomethane, 0.1-10% of sodium dodecyl sulfate and 1-1000 mmol/L of ethylene diamine tetraacetic acid;
a cell cross-linking solution; the cell crosslinking solution is a paraformaldehyde solution;
immunomagnetic beads; the immunomagnetic beads are protein A magnetic beads and/or protein G magnetic beads; and
diluting the solution; the diluent comprises the following components in molar concentration or mass percentage concentration: 0.01-1% of sodium dodecyl sulfate, 0.1-10% of polyethylene glycol octyl phenyl ether, 1-1000 mmol/L of ethylenediamine tetraacetic acid, 1-1000 mmol/L of tris (hydroxymethyl) aminomethane and 1-1000 mmol/L of sodium chloride.
2. The kit for co-immunoprecipitation recovery as recited in claim 1, wherein the wash solution comprises:
cleaning fluid A; the cleaning solution A comprises the following components in molar concentration or mass percentage concentration: 0.01-1% of sodium dodecyl sulfate, 0.1-10% of polyethylene glycol octyl phenyl ether, 1-1000 mmol/L of ethylenediamine tetraacetic acid, 1-1000 mmol/L of tris (hydroxymethyl) aminomethane and 1-1000 mmol/L of sodium chloride;
cleaning fluid B; the cleaning liquid B comprises the following components in percentage by mol or mass: 0.01-1% of sodium dodecyl sulfate, 0.1-10% of polyethylene glycol octyl phenyl ether, 1-1000 mmol/L of ethylenediamine tetraacetic acid, 1-1000 mmol/L of tris (hydroxymethyl) aminomethane and 1-2000 mmol/L of sodium chloride; and
cleaning fluid C; the cleaning solution C comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L lithium chloride, 0.1-10% ethyl phenyl polyethylene glycol, 0.1-10% sodium deoxycholate, 1-1000 mmol/L ethylene diamine tetraacetic acid and 1-1000 mmol/L tris (hydroxymethyl) aminomethane.
3. The kit for co-immunoprecipitation recovery of claim 1, wherein the eluate includes one or more of a DNA eluate, an RNA eluate, and a protein eluate.
4. The kit for co-immunoprecipitation recovery according to claim 3, wherein the DNA eluate comprises the following components in molar or percent by mass concentrations: 1-1000 mmol/L of trihydroxymethyl aminomethane, 0.1-10% of sodium dodecyl sulfate, 1-1000 mmol/L of magnesium chloride and 1-1000 mmol/L of sodium chloride;
the RNA eluent comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L of trihydroxymethyl aminomethane, 0.1-10% of sodium dodecyl sulfate, 0.1-10% of ethyl phenyl polyethylene glycol, 1-1000 mmol/L of magnesium chloride and 1-1000 mmol/L of sodium chloride;
the protein eluent comprises the following components in molar concentration or mass percentage concentration: 1-1000 mmol/L of trihydroxymethyl aminomethane, 1-1000 mmol/L of sodium chloride, 0.1-10% of ethyl phenyl polyethylene glycol, 0.1-10% of sodium deoxycholate, 0.1-10% of sodium dodecyl sulfate and 1-1000 mmol/L of ethylene diamine tetraacetic acid.
5. The kit for co-immunoprecipitation recovery according to claim 1, wherein the mass concentration of paraformaldehyde in the cell cross-linking solution is 0.1% to 10%.
6. Use of a kit according to any one of claims 1 to 5 for the interaction of proteins with cellular molecules.
7. A use as claimed in claim 6, wherein the cellular molecule is any one of DNA, RNA, protein and chromatin.
8. An application according to claim 7, characterized in that it comprises the following steps:
mixing the immunomagnetic beads and the diluent, then placing the mixture on a magnetic rack for separation, and removing supernatant to obtain a first precipitate;
mixing the first precipitate with the diluent, and then incubating with an antibody to obtain an incubation liquid;
separating the incubation liquid on a magnetic frame, and removing the supernatant to obtain a second precipitate for later use;
after culturing the cells, fixing the cells by using the cell cross-linking solution, and adding a glycine solution to neutralize the cell cross-linking solution;
mixing the fixed cells with the cell lysate, and then carrying out ultrasonic and centrifugal treatment to obtain a sample;
diluting a sample by using the diluent to obtain a diluted sample;
mixing the second precipitate with the diluted sample, and separating in a magnetic rack to obtain a third precipitate;
washing the third precipitate with the cleaning solution;
mixing the washed third precipitate with the eluent, adding or not adding protease, placing the mixture on a magnetic frame for separation, and collecting supernatant;
purifying the cell molecules in the supernatant.
9. Use of a kit according to any one of claims 1 to 5 in the manufacture of a kit for the treatment and/or prevention of a disease or epigenetic test.
10. A use according to claim 9, wherein the disease is any one of cancer, cardiovascular disease and central nervous system disorder.
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| CN113848102A (en) * | 2021-09-27 | 2021-12-28 | 深圳市心月生物科技有限公司 | Methylamphetamine extracting solution and preparation method thereof |
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