CN115754306A - Method for pulling down target protein by compound - Google Patents
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- CN115754306A CN115754306A CN202211539527.2A CN202211539527A CN115754306A CN 115754306 A CN115754306 A CN 115754306A CN 202211539527 A CN202211539527 A CN 202211539527A CN 115754306 A CN115754306 A CN 115754306A
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Abstract
The invention provides a method for pulling down a target protein by a compound, which comprises the steps of treating cells by using a lysate to obtain total cell protein, wherein the lysate comprises a protease inhibitor; pretreating the total cell protein by using a protein G/A magnetic bead to obtain the total cell protein to be detected so as to remove non-specifically combined protein in the total cell protein; pretreating avidin magnetic beads by using a biotin-labeled compound in a pull-down buffer solution containing bovine serum albumin to obtain avidin magnetic beads combined with the biotin-labeled compound; adding the pretreated avidin magnetic beads into the total protein of the cells to be tested for incubation treatment to obtain avidin magnetic beads capturing specific target proteins; and (3) treating the avidin magnetic beads obtained after incubation treatment by using biotin or a compound which is not marked by the biotin to obtain the target pull-down target protein.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for pulling down a target protein by a compound.
Background
Proteins are important components constituting all cells and tissues of a human body, are main players of cell functions, regulate functions such as transcription, translation, cell cycle, DNA replication and the like, and have important significance in maintaining normal cell functions, controlling communication among cells and allowing the cells to adapt to changes of environments. The important action way of the exogenous compound influencing the organism is generated by disturbing the dynamic regulation mechanism of the interaction network between biomolecules through the interaction with macromolecules such as protein and the like and then triggering a series of cascade responses such as subsequent cell signal conduction and multi-level key events such as cells, tissues, organs and the like. The research on the action target of the foreign compound protein can provide a breakthrough for the action mechanism of the foreign compound protein.
Among the increasingly prominent environmental problems, the screening of potential protein targets of pollutants in organisms is far behind the detection of toxic effects of the pollutants; in the research and development of new drugs, the search for unknown protein targets is an indispensable link for pharmacological and toxicological explanations in the research and development of drugs. The biotin-avidin system is a novel biological reaction amplification system developed in the late 70 s, and shows great potential in the exploration of target proteins of exogenous compounds due to the excellent sensitivity, specificity, stability and the like. However, the use of this system in compound-target protein interactions is limited, especially with the compound, the pull-down protein, which is difficult to achieve by itself, has a low success rate, and is difficult to enrich for sufficient amounts of protein for detection, thus limiting its use and making it somewhat user-friendly. At present, no rule for achieving uniform or optimal conditions exists, and an experimental scheme for the function of the method needs to be explored through a large number of experiments.
Disclosure of Invention
In view of the above, the main object of the present invention is to provide a method for down-drawing a target protein by a compound, which is expected to at least partially solve at least one of the above-mentioned technical problems.
In order to achieve the purpose, the technical scheme of the invention is as follows:
as one aspect of the present invention, there is provided a method for down-pulling a target protein from a compound, comprising the steps of: treating cells by using a lysate to obtain total cell protein, wherein the lysate comprises a protease inhibitor; pretreating the total cell protein by using a protein G/A magnetic bead to obtain the total cell protein to be detected so as to remove non-specifically bound protein in the total cell protein; pretreating avidin magnetic beads by using a biotin labeled compound in a drop-down buffer solution containing bovine serum albumin to obtain avidin magnetic beads combined with the biotin labeled compound, wherein the biotin labeled compound is a biotin labeled compound; adding the avidin magnetic beads obtained after pretreatment into the total cell protein to be tested for incubation treatment to obtain avidin magnetic beads capturing specific target protein; and (3) treating the avidin magnetic beads obtained after incubation treatment by using biotin or a compound which is not marked by the biotin to obtain the target pull-down target protein.
Based on the technical scheme, the method for pulling down the target protein by the compound at least has the following beneficial effects:
the invention provides a method for pulling down a target protein by a compound, which is based on a biotin avidin system, and comprises the steps of firstly obtaining total cell protein by using lysis solution containing a protease inhibitor, then removing non-specific binding protein in the total cell protein by using protein G/A magnetic beads, avoiding the non-specific binding of the non-specific protein to avidin beads, providing favorable conditions for the subsequent avidin beads to be coated with a biotin-labeled low-molecular compound to specifically capture the target protein, having high specificity, and then treating the biotin-labeled low-molecular compound and the avidin magnetic beads with a bovine serum albumin-containing pull-down buffer solution to specifically capture the target protein. The method has the advantages of high specificity of the captured target protein, short period and simple operation, and provides a new feasible method for the research of unknown targets of compounds.
Drawings
FIG. 1 is a block flow diagram of a method for down-drawing a target protein from a compound of the invention;
FIG. 2 is a passage chart of human induced pluripotent stem cells in an example of the present invention;
FIG. 3 is a diagram showing the growth state of human induced pluripotent stem cells according to an embodiment of the present invention;
FIG. 4 is a silver staining pattern of protein bands of examples and comparative examples of the present invention and protein standards.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
In the process of implementing the invention, it is found that how to implement the compound to pull down the target protein, and the amount of the protein which is enriched to be detected is the technical difficulty of the specific capture of the pull down protein target based on the biotin avidin system. The invention provides a method for pulling down a target protein by a compound, which is based on a biotin avidin system and comprises the steps of removing non-specific binding protein in total cell protein by using protein G/A magnetic beads, avoiding the non-specific binding of the non-specific protein to avidin beads, providing favorable conditions for the subsequent avidin beads to be coated with a biotin-labeled low-molecular compound to specifically capture the target protein, having high specificity, treating the biotin-labeled compound and the avidin magnetic bead specific capture target protein by using a pulling-down buffer solution containing bovine serum albumin, and further determining and optimizing which cell or tissue type and which lysis buffer solution are used, the time for incubating the beads with the lysis solution and the washing severity.
To further illustrate the present invention, preferred embodiments of the present invention are described below with reference to examples, but it should be understood that these descriptions are only for the purpose of further illustrating the features and advantages of the present invention, and are not intended to limit the claims of the present invention, and all other examples obtained by one of ordinary skill in the art without inventive faculty are within the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments of the methods and materials are described herein for illustrative purposes only. Moreover, descriptions of well-known techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
Specifically, according to some embodiments of the present invention, a method for pulling down a target protein by a compound is provided, comprising the following steps a to E (fig. 1).
Step A: and (3) treating the cells by using a lysate to obtain the total cell protein, wherein the lysate comprises a protease inhibitor.
According to the embodiment of the present invention, the obtaining of total cellular protein by treating cells with a lysate in this step is performed by the following steps A1 to A2.
In step A1, the cultured cells are collected by low temperature centrifugation, wherein the culture density of the cells is preferably equal to or more than 3.8X 10 5 Individual cell/cm 2 The cells include mammalian cells, preferably, human pluripotent stem cells
According to the embodiment of the invention, the cells with good growth vigor are taken out of the culture box, the culture medium is discarded, and phosphate buffer solution is added for shaking and cleaning; removing the phosphate buffer solution, adding a new phosphate buffer solution, hanging the cells from the bottom of the culture dish by using a cell scraper, and collecting the cells into a centrifuge tube; placing the cells in a low-temperature centrifuge, and centrifuging and collecting the cells; the centrifuged supernatant was discarded, and phosphate buffer was added again to resuspend the cells, followed by centrifugation again.
In step A2, the collected cells are lysed by using a lysis solution to obtain total cell protein, wherein the lysis time is 10 to 30 minutes.
According to the embodiment of the invention, the supernatant after centrifugation is discarded, RIPA lysate containing protease inhibitor is added, the mixture is blown and beaten uniformly, the mixture is placed on ice for lysis, the lysed cells are placed in a low-temperature centrifuge for high-speed centrifugation, and insoluble precipitates are removed; taking supernatant after centrifugation as total cell protein.
According to the embodiment of the present invention, the lysis time is too short (< 10 minutes), the cell lysis is not complete, the lysis time is too long (> 30 minutes), the protein is degraded, and the intact protein is not obtained, therefore, the lysis time is suitably in the range of 10 to 30 minutes, such as 10 minutes, 20 minutes, and 30 minutes.
According to an embodiment of the invention, protease inhibitors are added to the lysate to inhibit degradation of the protein, to ensure the integrity of the protein. In addition, the RIPA lysate containing the protease inhibitor is beneficial to fully lysing cell membrane protein, improving the total protein content and avoiding target point detection errors caused by protein collection loss.
According to the embodiment of the invention, the human induced pluripotent stem cells are closer to the species and the proportion of the protein in the development stage of the human early embryo, have good protein abundance and are beneficial to the detection of trace combined target protein in the process of pulling down the target protein by a compound.
And B: and (3) pretreating the total cell protein by using a protein G/A magnetic bead to obtain the total cell protein to be detected so as to remove the non-specifically bound protein in the total cell protein.
According to the embodiment of the invention, the step of pretreating the total cell protein by using the protein G/A magnetic beads to obtain the total cell protein to be detected is realized by the following steps B1-B2.
In step B1: and (3) diluting the total cell protein by using a pull-down buffer solution to obtain a dilution solution of the total cell protein.
Placing the pulling-down buffer solution on ice for precooling, taking the precooled pulling-down buffer solution, adding the pulling-down buffer solution into the total cell protein, blowing, sucking and uniformly mixing, placing the diluted total cell protein in a centrifuge at 4 ℃ to remove insoluble precipitates, taking supernatant after centrifugation, namely the dilution of the total cell protein, and placing the supernatant on ice for later use. Wherein the centrifugal rotating speed is 12000-14000 rpm, and the time is 25-35 minutes.
In step B2: and adding the diluent of the total cell protein into the cleaned protein G/A magnetic beads, and incubating in a low-temperature environment for 2-8 hours at a temperature of 4-8 ℃.
According to the embodiment of the present invention, the incubation period is too short (< 2 hours), the non-specifically bound proteins remaining in the total cellular proteins are not beneficial for the subsequent avidin magnetic beads to specifically capture the target proteins, and the incubation period is too long (> 8 hours) to ensure the integrity of the proteins, therefore, the suitable time range of the incubation period is 2-8 hours, such as 2 hours, 6 hours, 4 hours, and 8 hours.
According to the embodiment of the invention, the protein G/A magnetic beads are taken out of the centrifuge tube, the pull-down buffer solution is added, the upper part and the lower part are inverted for a plurality of times, the pull-down buffer solution is discarded, and the step is repeated for 2-3 times to obtain the cleaned protein G/A magnetic beads. And D, adding the dilution liquid of the total cellular proteins prepared in the step B1 into the washed protein G/A magnetic beads, and placing the mixture in a low-temperature environment for upside-down incubation to remove the non-specifically bound proteins in the total cellular proteins. After the incubation period is completed, the protein G/A magnetic beads are removed, and the supernatant is the total protein of the cell to be detected and is placed on ice for standby.
According to the embodiment of the invention, the protein G/A magnetic beads are utilized to remove non-specific binding protein in the total cell protein, so that the non-specific binding of the protein to the avidin beads is avoided, favorable conditions are provided for the subsequent avidin beads to be coated with a biotin-labeled low-molecular compound to specifically capture the target protein, and the target protein is favorably pulled down by the compound.
And C: and (2) pretreating the avidin magnetic beads by using a biotin labeled compound in a drop-down buffer solution containing bovine serum albumin to obtain avidin magnetic beads combined with the biotin labeled compound, wherein the biotin labeled compound is a biotin labeled compound.
According to the embodiment of the invention, the avidin magnetic beads are pretreated by using a biotin-labeled compound in a drop-down buffer solution containing bovine serum albumin, so that avidin magnetic beads combined with the biotin-labeled compound are obtained, and the method comprises the following steps: placing the avidin magnetic beads and the biotin labeled compound in a drop-down buffer solution containing bovine serum albumin for incubation treatment under a low temperature condition so as to coat the biotin labeled compound on the surface of the avidin magnetic beads, and simultaneously closing potential non-specific protein binding sites of the avidin magnetic beads to obtain the avidin magnetic beads combined with the biotin labeled compound, wherein the content percentage of the bovine serum albumin is 0.5-5%, the incubation treatment time is 3-8 hours, and the low temperature range is 4-8 ℃.
Further, placing the avidin magnetic beads and the biotin labeled compound in a pull-down buffer solution containing bovine serum albumin for incubation treatment at a low temperature to obtain the avidin magnetic beads combined with the biotin labeled compound, comprising: and (3) putting the avidin magnetic beads into a centrifugal tube, adding a pull-down buffer solution, inverting for a plurality of times, discarding the buffer solution, and repeating the step for 2-3 times to clean the avidin magnetic beads. And adding a pull-down buffer solution containing bovine serum albumin into the washed avidin magnetic beads, adding a biotin-labeled compound, placing the beads at a low temperature, inverting the beads upside down, incubating, and coating the biotin-labeled compound on the beads to obtain the avidin magnetic beads combined with the biotin-labeled compound. After the binding is completed, the buffer is discarded, the pull-down buffer is added, the upside down is inverted for several times, the pull-down buffer is discarded, the step is repeated for 2 to 3 times, and the beads are washed to remove the unbound biotin-labeled compound.
According to the embodiment of the invention, the incubation treatment time is too low (< 3 hours) and is not enough to obtain avidin magnetic beads with biotin-labeled compounds fully bound, and the incubation time is too high (> 8 hours) and is not more than capable of binding the biotin-labeled compounds on the avidin magnetic beads, so that the biotin-labeled compounds and the avidin magnetic beads are sufficiently bound within the incubation treatment time range, and the target protein is favorably pulled down by the compounds. Thus, suitable incubation times are in the range of 3 to 8 hours, for example 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours.
According to the embodiment of the invention, the bovine serum albumin is added into the pull-down buffer solution, so that the specificity of target protein capture is improved.
According to the embodiment of the invention, the loading capacity of the biotin labeled compound on the surface of the avidin magnetic bead can be flexibly adjusted.
Step D: and adding the avidin magnetic beads obtained after pretreatment into the total cell protein to be detected for incubation treatment to obtain avidin magnetic beads captured with specific target protein.
According to the embodiment of the invention, the avidin magnetic beads obtained after pretreatment are added into the total protein of the cells to be tested for incubation treatment, so as to obtain avidin magnetic beads captured with specific target protein, and the avidin magnetic beads comprise: placing the pretreated avidin magnetic beads into total cell protein to be detected, and placing the avidin magnetic beads in a low-temperature environment for incubation treatment so as to capture specific target protein corresponding to the compound, thereby obtaining avidin magnetic beads captured with the specific target protein, wherein the incubation treatment time is 2-10 hours, and the temperature range of the low-temperature environment is 4-8 ℃.
According to the embodiment of the invention, the incubation treatment time is within the range of 2-10 hours, such as 2 hours, 4 hours, 6 hours, 8 hours and 10 hours, so that the pretreated avidin magnetic beads can effectively capture the target protein, and meanwhile, the target protein is prevented from being degraded in the capture process, and the integrity of the target protein is protected.
Step E: and (3) treating the avidin magnetic beads obtained after incubation treatment by using biotin or a compound which is not marked by the biotin to obtain the target pull-down target protein.
According to the embodiment of the invention, avidin magnetic beads obtained after incubation treatment are treated by biotin or a compound which is not labeled by biotin, so as to obtain a target pull-down target protein, and the method comprises the following steps: and mixing biotin or a compound which is not marked by the biotin and an avidin magnetic bead body obtained after incubation treatment in a pull-down buffer solution, placing the mixture on ice for flicking and uniformly mixing to complete competitive elution, and collecting supernatant to obtain the target pull-down target protein, wherein the treatment time is 20-40 minutes, such as 20 minutes, 30 minutes and 40 minutes.
According to the embodiment of the invention, competitive elution is carried out by adopting biotin or a compound which is not marked by the biotin, so that false positive targets caused by combination of the biotin and an intermediate-linked compound and protein are reduced, and identification of the targets is more specific.
According to the embodiment of the invention, the method further comprises analyzing the target pull-down target protein to determine the type of the target pull-down target protein, and specifically comprises the steps F and G.
Step F: and separating and eluting the target pull-down target protein by utilizing polyacrylamide gel electrophoresis to obtain a target pull-down target protein band, and realizing the visualization of the target pull-down target protein band based on silver staining detection.
Step G: and detecting the target pull-down target protein band by using a liquid chromatography-mass spectrometer to determine the type of the target pull-down target protein.
According to the embodiment of the invention, in the step F, silver staining detection can quickly and preliminarily judge whether the target protein is fished out, so that the result is preliminarily judged before the liquid chromatography-mass spectrometry detection is carried out in the step G, and unnecessary subsequent experiments are avoided. In addition, in step F, silver staining detection can detect and retain extremely minute amounts of target pull-down target protein with high sensitivity, thus allowing a wider detection range of target pull-down target protein.
According to an embodiment of the invention, the pull-down buffer comprises HEPES, glycerol, KCl, mgCl 2 And EDTA. Wherein, the concentration of HEPES is 5-20 mM, the volume percentage of glycerol is 15-30%, the concentration of KCl is 95-105mM 2 The concentration of (A) is 1 to 2mM, and the concentration of EDTA is 0.1 to 0.3mM.
According to the embodiment of the invention, HEPES with corresponding concentration provides a buffering environment for protein, and KCl concentration is set to be beneficial to reducing non-specific binding background and ensuring the binding of target protein; the addition of glycerol in a corresponding proportion ensures the binding efficiency of the target protein while protecting the protein; the addition of MgCl2 and EDTA in corresponding proportion ensures the activity of the protein.
According to an embodiment of the present invention, the compound includes a low molecular weight compound having a molecular weight of 400 to 1500; the types of the low molecular weight compounds include perfluorosulfonic acid compounds, perfluorocarboxylic acid compounds, bisphenol A and inhibitor JQ1. Wherein the inhibitor JQ1 is tert-butyl (S) - (+) -2- (4- (4-chlorophenyl) -2,3,9-trimethyl-6H-thieno [3,2-F ] [1,2,4] triazolo [4,3-A ] [1,4] diazepin-6-yl) acetate.
According to an embodiment of the present invention, the avidin magnetic beads include streptavidin magnetic beads.
According to the embodiment of the invention, the streptavidin magnetic beads are more easily combined with biotin and are firmly combined, so that a basis is provided for realizing the pull-down of target protein by the compound.
According to the embodiment of the invention, in the step B, the protein G/A magnetic beads are used for removing the protein which is not specifically bound in the total cell protein, so that the protein which is not specifically bound in the total cell protein is prevented from being bound to the surfaces of the avidin magnetic beads, and favorable capture conditions are provided for the step D.
According to the embodiment of the invention, the implementation process of the steps A to E does not need a high coupling procedure method depending on instruments, and the method is economical and easy to popularize.
The technical solution of the present invention will be described in detail below by referring to a plurality of specific examples. It should be noted that the following specific examples are only for illustration and are not intended to limit the invention.
In the following examples, some reagents and detection instruments are described below:
in the following examples, human pluripotent induced stem cells (ipscs) were derived from the stem cell technology platform of the chinese academy of sciences cell bank; protein G magnetic beads, streptavidin magnetic beads, and protein standards were purchased from Saimer Feishell science and technology (China) Co., ltd.; RIPA lysate, KCl, mgCl 2 Purchased from Beijing Solaibao technologies, inc.; HEPES, glycerol, and other unrecited agents and drugs are readily available from Saimer Feishell science, inc. (China) or are prepared in the laboratory using current methods.
Examples
1. Obtaining of Total cellular protein
(1) One 6 cm dish of well-grown human Induced Pluripotent Stem Cells (iPSCs) was passaged to four 10 cm dishes, one of which was iPSCs as shown in FIG. 2, using mTeSR TM Plus was cultured for 4 days to obtain a culture density of 3.8X 10 5 Individual cell/cm 2 The growth state of the iPSC of (3) is shown in fig. 3.
(2) The cultured cells were taken out from the 37 ℃ incubator, the medium was discarded, and 5mL of phosphate buffer was added to shake and wash.
(3) The phosphate buffer was discarded, a new 5mL of phosphate buffer was added, and the cells were scraped from the bottom of the dish using a cell scraper and collected into a centrifuge tube.
(4) The cells were centrifuged at 2000rpm for 5 minutes in a 4 ℃ centrifuge to collect the cells.
(5) The centrifuged supernatant was discarded, and 10mL of phosphate buffer was added again to resuspend the cells, followed by centrifugation again.
(6) The centrifuged supernatant was discarded, and 1.5mL of RIPA lysate (containing 1 × pre-transcriptional complex (PIC), 1mM phenylmethanesulfonyl fluoride (PMSF), 2mM Dithiothreitol (DTT)) was added thereto, mixed by pipetting, and then lysed on ice for 10 to 30 minutes.
(7) The lysed cells were placed in a 4 ℃ centrifuge at 13000rpm for 30 minutes to remove insoluble pellet.
(8) Taking supernatant after centrifugation as total cell protein.
2. Pretreatment of total cellular protein
(1) Preparing a pull-down buffer solution, which comprises the following components: 20mM HEPES,20% glycerol, 100mM KCl,1.5mM MgCl 2 0.2mM EDTA, and placed on ice for precooling.
(2) Adding 3mL of precooled pulldown buffer into the total cell protein, and uniformly mixing by blowing and sucking.
(3) The diluted total cellular protein was centrifuged at 13000rpm for 30 minutes in a centrifuge at 4 ℃ to remove insoluble precipitates.
(4) Taking supernatant after centrifugation as cell total protein diluent, and placing on ice for standby.
(5) mu.L of the tip was subtracted, and 100. Mu.L of protein G magnetic beads were aspirated and placed in a 1.5mL centrifuge tube.
(6) Add pre-cooled pulldown buffer, reverse up and down for 5 minutes, discard buffer using magnetic stand, and repeat this step 2 times.
(7) And adding the prepared dilution solution of the total cellular proteins into the washed protein G magnetic beads, placing the mixture at 4 ℃ for incubation for 4 hours in an upside-down manner, and removing the non-specifically bound proteins in the total cellular proteins.
(8) After the incubation was not completed, the magnetic beads were removed using a magnetic rack, and the supernatant was the total cell protein after pretreatment and was placed on ice for use.
3. Pretreatment of streptavidin magnetic beads
(1) The tip of the 200. Mu.L tip was subtracted, and 100. Mu.L of streptavidin magnetic beads were each pipetted into two 1.5mL centrifuge tubes.
(2) To the removed streptavidin magnetic beads, pull-down buffer was added, turned upside down for 5 minutes, the buffer was discarded, and this step was repeated 2 times.
(3) Adding 1mL of pulldown buffer containing 0.5% bovine serum albumin into the washed streptavidin magnetic beads, and adding biotin-labeled perfluorooctane sulfonate with the working concentration of 200 mu M, wherein the biotin-labeled perfluorooctane sulfonate has a structure shown as a formula (1) and has the molecular weight of 928.7.
(4) And (3) placing the streptavidin magnetic beads at 4 ℃ and turning the streptavidin magnetic beads upside down, mixing the streptavidin magnetic beads evenly, and incubating the streptavidin magnetic beads for 3 to 8 hours.
(5) After the binding is finished, removing the buffer solution in the streptavidin magnetic beads, adding the precooled pull-down buffer solution, reversing the buffer solution up and down for 5 minutes, removing the buffer solution, repeating the step for 2-3 times, and cleaning the free biotin-labeled perfluorooctane sulfonic acid which is not bound to the streptavidin magnetic beads.
4. Obtaining specific target protein of perfluorooctane sulfonate
(1) And adding the total cell protein after pretreatment to streptavidin magnetic beads after pretreatment.
(2) And (3) placing the mixture of the streptavidin magnetic beads and the pretreated total cell protein at 4 ℃, and incubating for 3 hours in an upside-down manner to perform specific pull-down target protein capture.
(3) After the capture is completed, the supernatant is discarded by a magnetic frame, and streptavidin magnetic beads are collected.
(4) And adding a pull-down buffer solution into the coated streptavidin magnetic beads, reversing the coated streptavidin magnetic beads for 5 minutes, discarding the buffer solution, and repeating the step for 2-3 times.
(5) And adding 30 mu L of pull-down buffer solution into the washed magnetic beads, adding 40mM of biotin-unlabelled perfluorooctane sulfonate, gently blowing, uniformly mixing, placing on ice for 20-40 minutes, performing competitive elution, and gently flicking and uniformly mixing the mixture in the period to prevent the magnetic beads from precipitating.
(6) And (5) repeating the step (5) for 1-2 times to collect the streptavidin magnetic beads captured with the specific target protein as much as possible.
(7) And collecting the streptavidin magnetic beads captured with the specific target protein by a magnetic frame, eluting and collecting supernatant to obtain the perfluorooctane sulfonate specific target protein solution.
Comparative example
Similar to the system and procedure of example 1, except that in the step of pre-treating streptavidin magnetic beads (3), biotin was added at a working concentration of 200. Mu.M; in the step (5) of capturing the target protein, 40mM biotin is added, and the mixture is gently blown and beaten uniformly, so as to obtain a mixed solution in the step (7).
The solutions obtained in examples and comparative examples and the protein standard were separated and eluted by polyacrylamide gel electrophoresis to obtain protein bands, which were stained by silver staining, and the results after staining are shown in fig. 4.
As can be seen from fig. 4, no protein band is shown in the comparative example, but a protein band appears in the example, which illustrates that the perfluorooctane sulfonate pull-down specific target protein is successfully achieved by the operation flow of the method provided by the present invention, and further illustrates that the whole flow is optimized by using the protein G/a magnetic bead and the avidin magnetic bead together based on the biotin avidin system to achieve the pull-down of the protein by the compound, which has the advantages of high specificity, short cycle, and simple operation, and provides a new feasible method for the research of unknown target of the compound.
The above embodiments are provided to further explain the objects, technical solutions and advantages of the present invention in detail, and it should be understood that the above embodiments are only examples of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of compound pull-down of a target protein comprising the steps of:
treating cells with a lysis solution to obtain total cell protein, wherein the lysis solution comprises a protease inhibitor;
pretreating the total cell protein by using protein G/A magnetic beads to obtain the total cell protein to be detected so as to remove non-specifically bound protein in the total cell protein;
pretreating avidin magnetic beads by using a biotin-labeled compound in a drop-down buffer solution containing bovine serum albumin to obtain avidin magnetic beads combined with the biotin-labeled compound, wherein the biotin-labeled compound is the compound labeled by biotin;
adding the avidin magnetic beads obtained after pretreatment into the total protein of the cells to be tested for incubation treatment to obtain avidin magnetic beads captured with specific target proteins;
and (3) processing the avidin magnetic beads obtained after incubation treatment by using the biotin or the compound which is not marked by the biotin to obtain the target pull-down target protein.
2. The method of claim 1, wherein the step of treating the cell with the lysing solution to obtain the total cell protein comprises:
collecting the cultured cells by using a low-temperature centrifugation method, wherein the culture density of the cells is preferably more than or equal to 3.8 multiplied by 10 5 Individual cell/cm 2 The cells include mammalian cells, preferably, human pluripotent induced stem cells;
and cracking the collected cells by using the lysate to obtain the total cell protein, wherein the cracking time is 10-30 minutes.
3. The method of claim 1, wherein the pre-treating of the total cell protein with protein G/a magnetic beads to obtain the total cell protein to be tested comprises:
diluting the total cell protein by using a pull-down buffer solution to obtain a diluent of the total cell protein;
and adding the diluent of the total cell protein into the washed protein G/A magnetic beads, and incubating in a low-temperature environment, wherein the incubation time is 2-8 hours, and the temperature range of the low-temperature environment is 4-8 ℃.
4. The method of claim 1, wherein pretreating the avidin magnetic beads with a biotin labeling compound in a pulldown buffer containing bovine serum albumin to obtain avidin magnetic beads having the biotin labeling compound bound thereto comprises:
placing the avidin magnetic beads and the biotin labeling compound in a drop-down buffer solution containing bovine serum albumin for incubation treatment under a low temperature condition so as to coat the biotin labeling compound on the surface of the avidin magnetic beads, and simultaneously closing potential non-specific protein binding sites of the avidin magnetic beads to obtain the avidin magnetic beads combined with the biotin labeling compound, wherein the content percentage of the bovine serum albumin is 0.5-5%, the incubation treatment time is 3-8 hours, and the low temperature range is 4-8 ℃.
5. The method of claim 4, wherein the draw down buffer comprises HEPES, glycerol, KCl, mgCl 2 And EDTA. Wherein, the concentration of HEPES is 5-20 mM, the volume percentage of glycerol is 15-30%, the concentration of KCl is 95-105mM 2 The concentration of (A) is 1 to 2mM, the concentration of EDTA is 0.1 to 0.3mM。
6. The method according to claim 1, wherein the avidin magnetic beads obtained after the pretreatment are added to the total protein of the cells to be tested for incubation, so as to obtain avidin magnetic beads with captured specific target protein, comprising:
placing the pretreated avidin magnetic beads into the total cell protein to be detected, and placing the avidin magnetic beads in a low-temperature environment for incubation treatment so as to capture specific target proteins corresponding to the compounds, so as to obtain avidin magnetic beads with the captured specific target proteins, wherein the incubation treatment time is 2-10 hours, and the temperature range of the low-temperature environment is 4-8 ℃.
7. The method according to claim 1, wherein the treating of the avidin magnetic beads obtained after the incubation with the biotin or the compound not labeled with biotin to obtain a target pull-down target protein comprises:
and mixing the biotin or the compound which is not marked by the biotin and the avidin magnetic beads obtained after incubation treatment in a pull-down buffer solution, placing the mixture on ice for flicking and uniformly mixing to complete competitive elution, and collecting supernatant to obtain the target pull-down target protein, wherein the treatment time is 20-40 minutes.
8. The method of claim 1, further comprising:
analyzing the target pull-down target protein to determine the type of the target pull-down target protein, wherein the method comprises the following steps:
separating and eluting the target pull-down target protein by utilizing polyacrylamide gel electrophoresis to obtain a target pull-down target protein band, realizing the visualization of the target pull-down target protein band based on silver staining detection, and
and detecting the target pull-down target protein band by using a liquid chromatography-mass spectrometer to determine the type of the target pull-down target protein.
9. The method of claim 1, wherein the compound comprises a low molecular weight compound having a molecular weight of 400 to 1500; the types of the low molecular weight compounds comprise perfluorosulfonic acid compounds, perfluorocarboxylic acid compounds, bisphenol A and an inhibitor JQ1.
10. The method of claim 4, wherein the avidin magnetic beads comprise streptavidin magnetic beads.
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| CN116482345B (en) * | 2023-06-16 | 2023-09-22 | 军科正源（北京）药物研究有限责任公司 | Method for detecting occupancy of dual-target drug receptor |
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