CN109517029B - Method for effectively fishing rice promoter DNA binding protein - Google Patents
Method for effectively fishing rice promoter DNA binding protein Download PDFInfo
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- CN109517029B CN109517029B CN201811420354.6A CN201811420354A CN109517029B CN 109517029 B CN109517029 B CN 109517029B CN 201811420354 A CN201811420354 A CN 201811420354A CN 109517029 B CN109517029 B CN 109517029B
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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Abstract
The invention provides a method for effectively fishing rice promoter DNA binding protein, which comprises the steps of extracting natural total protein of rice leaves by using a protein extraction buffer solution containing NP-40, NaCl, PMSF and the like, further labeling a primer by using biotin and amplifying the promoter DNA, incubating the promoter DNA with magnetic beads coupled with streptavidin, and incubating a compound of the promoter DNA and the magnetic beads and the natural total protein of the rice leaves on the basis to obtain the protein combined with the promoter DNA. The method is suitable for researching the promoter DNA binding protein of different varieties of rice and can also be used for researching the binding protein of promoter DNA of different plants. Establishes a technology for efficiently and simply obtaining the plant promoter DNA binding protein.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for effectively fishing a rice promoter DNA binding protein.
Background
Gene expression is the fundamental process by which an organism performs vital activities and is the initial change in the organism's response. In organisms, gene expression is determined by the action of trans-acting factors (transcription factors) on cis-acting elements (promoter regions) in organisms, the surface level of a gene is closely related to the activity of the promoter, and the activity of the promoter is regulated by the transcription factors. The transcription factor is a trans-acting factor, and plays an important role in the growth and development of animals and plants and the response of the animals and the plants to the external environment.
In higher plants, transcription factors not only participate in the regulation of important processes in life activities such as growth and development of plants and morphogenesis, but also regulate the processes of secondary metabolism, stress-resistant reaction and the like of plants. In addition, the protein encoded by a plurality of genes in the genes capable of encoding the proteins in the plants is transcription factor protein, and the number and the variety of the transcription factors are various, thereby indicating the specificity and the complexity of plant transcription regulation. Therefore, the research on the transcription factors of the gene promoters of higher plants under different environmental conditions has very important significance for revealing the transcription regulation process of plant gene response.
In response, obtaining the transcription factor of the target gene promoter has an important role in researching the expression regulation behavior of the target gene. Yeast monohybrid is a classical method for screening transcription factors having specific binding regions to target elements, which is capable of efficiently detecting specific interactions between specific transcription factors and cis-acting elements. However, the method needs to construct fusion expression vectors of different target genes and transcriptional Activation regions (Activation domain AD), the process is complicated, the screening of positive interactors is also complicated, the requirement on operation conditions is high, and the wide application of the method is limited. Therefore, there is a need to develop a method for efficiently obtaining a transcription factor bound to a target promoter DNA.
Disclosure of Invention
The invention provides a method for effectively fishing rice promoter DNA binding protein, which can obtain a transcription factor bound on a target promoter DNA at one time and can be used for researching an expression regulation factor of a target gene.
A method for effectively fishing a rice promoter DNA binding protein comprises the following steps:
(1) extracting natural total protein of rice;
(2) amplifying promoter DNA and marking biotin;
(3) incubating promoter DNA marked by biotin and streptavidin coupled magnetic beads;
(4) binding protein of promoter DNA is fished and separated.
The specific steps of the step (1) of extracting the natural total protein of the rice are as follows: putting 3-5 g of fresh rice leaves into a precooled mortar, and grinding the fresh rice leaves into powder under the condition of liquid nitrogen; and (2) quickly transferring the powder into a pre-cooled 50 ml centrifuge tube, adding a protein extraction buffer solution with the volume of 1.5-2 times that of the tissue powder, fully and uniformly mixing by vortex oscillation, carrying out ice bath for 15-30 min, carrying out rotary incubation for 45-60 min at 4 ℃, centrifuging for 15-20 min at 4 ℃ at 15000g, taking the supernatant, filtering residues in the supernatant by using sterilized Miracloth, further centrifuging for 15-20 min at 4 ℃ at 15000g, filtering a small amount of residues by using the sterilized Miracloth again, finally obtaining a rice natural total protein solution, and storing for later use at 4 ℃.
The specific method for amplifying the promoter DNA and labeling the biotin in the step (2) comprises the following steps: designing forward and reverse amplification primers aiming at a researched target promoter, wherein biotin (biotin) labeling is carried out on the 5' end of the forward primer, the promoter DNA with the biotin label is obtained by amplification by taking rice DNA as a template, and the promoter DNA is recovered and purified by agarose gel electrophoresis and stored at-20 ℃ for later use.
The target promoter isPALA promoter, wherein the forward primer sequence is: 5'-GGTAGTGCTAGATATGAAGGGCTGC-3', reverse primer sequence 5'-GCACGAGGAGAAGAGAGGATTCGAT-3'.
The specific operation steps of the biotin-labeled promoter DNA and streptavidin coupled magnetic bead incubation in the step (3) are as follows: taking 15-20 mu L of streptavidin coupled magnetic beads to a 1.5 ml sterile centrifuge tube, placing the centrifuge tube in a magnetic frame, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking off a supernatant; taking out the centrifuge tube, adding 60-80 mu L Binding solution to lightly resuspend the magnetic beads, putting the centrifuge tube in a magnetic frame again after resuspension, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking off the supernatant; taking out the centrifuge tube, adding 60-80 mu L of Binding solution to lightly resuspend the magnetic beads, adding the biotin labeled promoter DNA with the same volume and the total volume of 10-15 pmole, lightly mixing, placing in a rotary mixer, and incubating for 3-4 h at room temperature; after incubation, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2-3 min, storing the centrifugal tube in the magnetic frame, and carefully sucking off a supernatant; taking out the centrifuge tube, adding 120-160 mu L of Washing solution for gently resuspending the magnetic beads, putting the centrifuge tube in a magnetic frame again after resuspension, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking off the supernatant; taking out the centrifuge tube, adding 120-160 mu L of sterile double distilled water to lightly resuspend the magnetic beads, putting the centrifuge tube into the magnetic frame again after resuspension, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking the supernatant; taking out the centrifuge tube, and resuspending the magnetic beads by using 15-20 mu L of protein extraction buffer solution; wherein the Binding solution and Washing solution are selected from Dynabeads kinase B DNA Kit of Saimer Feishale technologies, Inc.
The specific operation steps of fishing and separating the binding protein of the promoter DNA in the step (4) are as follows: adding the magnetic beads containing the biotin-labeled promoter DNA re-suspended in the step (3) into the natural total protein solution of the rice obtained in the step (1), placing the mixture into a rotary mixer, and incubating the mixture for 3-4 hours at 4 ℃; after incubation, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2-3 min, storing the centrifugal tube in the magnetic frame, and carefully sucking off a supernatant; washing the magnetic beads for 2 times by using 120-160 mu L of protein extraction buffer solution, and keeping the centrifugal tube in a magnetic frame in the process; and after washing, taking out the centrifuge tube, adding 15-20 mu L of 2 xSDS buffer for resuspending magnetic beads, cooking at 100 ℃ for 8-10 min, taking out, standing to room temperature, placing in a magnetic frame, standing for 2-3 min, slowly sucking out supernatant to obtain a solution of promoter DNA binding protein, and separating the protein by adopting 10% v/v polyacrylamide gel electrophoresis.
The 2 x SDS buffer contained the following final concentrations of components: 20% v/v glycerol, 0.02% w/v bromophenol blue, 4% w/v SDS, 0.2M Tris. Cl pH 6.8, 200 mM DTT.
The protein extraction buffer contained the following final concentrations of components: 50 mM Tris & Cl pH 7.5, 150 mM NaCl, 1mM EDTA pH 8.0, 0.1% v/v Nonidet P-40, 1% v/v Triton X-100, 1mM PMSF, 1 xcOmplete Protease Inhibitor cocktail。
The method for obtaining the binding protein of the rice promoter DNA comprises the steps of firstly, quickly grinding a tissue sample of rice into powder by using liquid nitrogen in a mortar, further transferring the sample powder into a 50 ml centrifugal tube, adding lysate with 2 times of volume, uniformly mixing by vortex, carrying out ice bath, carrying out rotary incubation and extracting protein (figure 1), further carrying out amplification and biotin labeling on target promoter DNA (figure 2), then carrying out incubation on the target promoter DNA and streptavidin coupled magnetic beads, further carrying out incubation on the target promoter DNA and the streptavidin coupled magnetic beads containing the promoter and protein solution, and collecting the protein bound on the promoter DNA through the attraction effect of a magnetic frame after further incubation. After SDS-PAGE electrophoretic separation and Coomassie brilliant blue solution staining of the protein, the result can see obvious protein bands, and the result is shown in figure 3 and is used for mass spectrum identification; it can be seen that the method of the present invention can effectively fish proteins bound to promoter DNA. Repeated tests prove that the protein combined on the promoter DNA, which is fished by the method, has good repeatability and high reliability, and can be efficiently used for researching the expression regulatory factor of the gene.
The invention has the following remarkable advantages:
(1) the method is suitable for different rice varieties, can be applied to the research of plants such as arabidopsis thaliana and tobacco, and has wide applicability;
(2) the reproducibility is good, and the reliability is high;
(3) the extraction is convenient, a complex treatment process is not needed, and the extraction conditions are not strict;
(4) the transcription factor bound to the promoter DNA can be obtained in a short time.
Drawings
FIG. 1 shows the results of electrophoresis of the natural proteins of leaves of rice PI312777 and Lemont; wherein lanes 1-3 are all natural protein of leaf of rice PI 312777; lanes 4-6 are rice Lemont leaf native total protein.
FIG. 2 shows the biotin labeling of rice PI312777 and LemontPALElectrophoresis detection results of the gene promoter; wherein lanes 1-4 are all PAL gene promoters of rice PI 312777; lane 5-8 shows the rice gene PAL promoter of Lemont.
FIG. 3 shows the growth of rice PI312777 and LemontPALDetecting the binding protein of the gene promoter by electrophoresis; wherein the arrow in lane 3 is that of rice LemontPALAnd lane 4 is a negative control of the combination of the natural total protein of rice Lemont leaf and streptavidin coupled magnetic beads.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1
The method for obtaining the binding protein of the rice promoter DNA comprises the following specific steps:
(1) raw materials: leaf of rice variety PI312777 from Fujian province (Fujian university of agriculture and forestry teaching farm greenhouse).
(2) Extraction of natural total protein of rice PI312777 leaf: taking 4 g of fresh leaves of the rice PI312777, putting the leaves into a precooled mortar, and quickly grinding the leaves into powder under the condition of liquid nitrogen; the powder was quickly transferred to a pre-cooled 50 ml centrifuge tube and 30 ml of extraction buffer (50 mM Tris. Cl pH 7.5; 150 mM NaCl; 1mM EDTA pH 8.0; 0.1% v/v Nonidet P-40; 1% v/v Triton X-100; 1mM PMSF; 1 xcOApple Protease Inhibitor cocktail, Roche), vortexing, mixing, ice-cooling for 20 min, rotary incubating at 4 deg.C for 60 min, centrifuging at 4 deg.C for 15 min at 15000g, collecting supernatant, filtering with sterilized miracloth (Millipore), and collecting supernatantThe residue and the filtered supernatant were further centrifuged at 15000g at 4 ℃ for 15 min, and a small amount of the residue was filtered again with sterilized miracloth (Millipore) to obtain a solution of rice total protein in nature, which was then electrophoretically detected (FIG. 1, lanes 1-3) and stored at 4 ℃ until use.
（3）PALAmplification and labeling of promoter DNA: by usingPALA forward and reverse amplification primer (the forward primer sequence is F: 5'-GGTAGTGCTAGATATGAAGGGCTGC-3', and the reverse primer sequence is R: 5'-GCACGAGGAGAAGAGAGGATTCGAT-3') is designed for a gene promoter, biotin (biotin) labeling is carried out at the 5' end of the forward primer, the promoter DNA with the biotin label is obtained by amplification by taking the genome DNA of the rice PI312777 as a template, and the promoter DNA is recovered and purified by agarose gel electrophoresis and stored at-20 ℃ for later use. Biotin-labelledPALThe results of the electrophoretic detection of the gene promoter are shown in FIG. 2 (lanes 1-4).
（4）PALBinding promoter DNA with streptavidin-coupled magnetic beads: 20 μ L of Streptavidin-coupled magnetic beads (Streptavidin-conjugated Dynabeads, Dynabeads) were taken®kilobasetBinder Kit, Invitrogen) in a 1.5 ml sterile centrifuge tube, placing the centrifuge tube in a magnetic frame, standing for 2min, storing the centrifuge tube in the magnetic frame, carefully removing the supernatant; taking out the centrifuge tube, adding 80 μ L Binding solution to lightly resuspend the magnetic beads, placing the centrifuge tube in the magnetic frame again after resuspension, standing for 2min, storing the centrifuge tube in the magnetic frame, and carefully sucking the supernatant; and (3) taking out the centrifuge tube, adding 80 mu L Binding solution to lightly resuspend the magnetic beads, adding the biotin labeled promoter DNA with the same volume and the total amount of 15 pmole, lightly mixing uniformly, placing in a rotary mixer, and incubating for 3-4 h at room temperature. After the incubation is finished, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2min, storing the centrifugal tube in the magnetic frame, and carefully sucking the supernatant; taking out the centrifuge tube, adding 160 mu L of Washing solution to lightly resuspend the magnetic beads, putting the centrifuge tube in the magnetic frame again after resuspension, standing for 2min, storing the centrifuge tube in the magnetic frame, and carefully sucking the supernatant; taking out the centrifuge tube, adding 160 μ L sterile double distilled water to lightly resuspend the magnetic beads, placing the centrifuge tube in the magnetic frame again after resuspension for 2min, storing the centrifuge tube in the magnetic frame, carefully suckingRemoving the supernatant; the centrifuge tube was removed and the beads were washed with 20. mu.L of protein extraction buffer (50 mM Tris. Cl pH 7.5; 150 mM NaCl; 1mM EDTA pH 8.0; 0.1% v/v Nonidet P-40; 1% v/v Triton X-100; 1mM PMSF; 1 xcOComplete Protease Inhibitor cocktail, Roche).
（5）PALBinding protein of promoter DNA fishing and separation: resuspending the protein containing the biotin labelPALThe magnetic beads of the promoter DNA are added into the natural total protein solution of the rice PI312777 and then placed into a rotary mixer, and incubated for 3h at 4 ℃. After the incubation is finished, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2min, storing the centrifugal tube in the magnetic frame, and carefully sucking the supernatant; mu.L of protein extraction buffer (50 mM Tris. Cl pH 7.5; 150 mM NaCl; 1mM EDTA pH 8.0; 0.1% v/v Nonidet P-40; 1% v/v Triton X-100; 1mM PMSF; 1 xcOComplete Protease Inhibitor cocktail, Roche) washed the beads 2 times, which keeps the centrifuge tubes in the magnetic rack. After washing, taking out the centrifuge tube, adding 20 μ L of 2 xSDS buffer to resuspend the magnetic beads, cooking at 100 ℃ for 8 min, taking out, standing to room temperature, placing in a magnetic frame, standing for 2min, slowly sucking out the supernatant to obtain a solution of promoter DNA binding protein, separating the protein by 10% v/v polyacrylamide gel electrophoresis, and obtaining the result shown in figure 3, wherein the arrow in the lane 1 is that of the rice PI312777PALThe binding protein of the gene promoter, lane 2 is a negative control of the combination of the natural total protein of the leaf of the rice PI312777 and the streptavidin coupled magnetic beads, and the result shows that the protein contains the rice PI312777PALThe magnetic beads of the gene promoter can effectively fish the protein bound to the promoter DNA.
Example 2
(1) Raw materials: the rice variety Lemont leaf, from fok-building fuzhou (teaching farm greenhouse at the university of fok-building agriculture and forestry).
(2) Extraction of natural total protein from rice Lemont leaves: taking 3 g of rice Lemont fresh leaves in a precooled mortar, and quickly grinding the rice Lemont fresh leaves into powder under the condition of liquid nitrogen; the powder was quickly transferred to a pre-chilled 50 ml centrifuge tube and 20 ml of extraction buffer (50 mM Tris. Cl pH 7.5; 150 mM NaCl; 1mM EDTA pH 8.0; 0.1% v/v Nonidet P-40; 1% v/v Triton X-100; 1mM PMSF; 1 ml Chun ShuzocOComplete mixing by vortex, ice bath for 20 min, rotary incubation for 60 min at 4 ℃, centrifugation for 15 min at 15000g at 4 ℃, supernatant filtration of residues in supernatant by sterilized miracloth (Millipore), centrifugation of filtered supernatant for 15 min at 4 ℃ at 15000g, and secondary filtration of residues by sterilized miracloth (Millipore) to obtain natural total protein solution of rice, electrophoresis detection of the obtained natural total protein solution (figure 1, lanes 4-6), and storage at 4 ℃ for standby.
（3）PALAmplification and labeling of promoter DNA: by usingPALA forward and reverse amplification primer (the forward primer sequence is F: 5'-GGTAGTGCTAGATATGAAGGGCTGC-3', and the reverse primer sequence is R: 5'-GCACGAGGAGAAGAGAGGATTCGAT-3') is designed for the promoter of the gene, wherein biotin (biotin) labeling is carried out at the 5' end of the forward primer, the promoter DNA with the biotin label is obtained by amplification by taking the genome DNA of rice Lemont as a template, and the promoter DNA is recovered and purified by agarose gel electrophoresis and stored at-20 ℃ for later use. Biotin-labelledPALThe results of the electrophoretic detection of the gene promoter are shown in FIG. 2 (lanes 5-8).
（4）PALBinding promoter DNA with streptavidin-coupled magnetic beads: mu.L of Streptavidin-coupled magnetic beads (Streptavidin-conjugated Dynabeads, Dynabeads) was taken®kilobasetBinder Kit, Invitrogen) in a 1.5 ml sterile centrifuge tube, placing the centrifuge tube in a magnetic frame, standing for 2min, storing the centrifuge tube in the magnetic frame, carefully removing the supernatant; taking out the centrifuge tube, adding 60 μ L Binding solution to lightly resuspend the magnetic beads, placing the centrifuge tube in the magnetic frame again after resuspension, standing for 2min, storing the centrifuge tube in the magnetic frame, and carefully sucking the supernatant; taking out the centrifuge tube, adding 60 mu L Binding solution to lightly resuspend the magnetic beads, adding the biotin labeled promoter DNA with the same volume and the total volume of 10 pmole, gently mixing the mixture, placing the mixture in a rotary mixer, and incubating the mixture at room temperature for 3 h. After the incubation is finished, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2min, storing the centrifugal tube in the magnetic frame, and carefully sucking the supernatant; taking out the centrifuge tube, adding 120 μ L Washing solution to lightly resuspend the magnetic beads, placing the centrifuge tube in the magnetic frame again after resuspension for 2min, and storing the centrifuge tubeCarefully blotting off the supernatant in a magnetic rack; taking out the centrifuge tube, adding 120 mu L of sterile double distilled water to lightly resuspend the magnetic beads, putting the centrifuge tube in the magnetic frame again after resuspension, standing for 2min, storing the centrifuge tube in the magnetic frame, and carefully sucking the supernatant; the centrifuge tube was removed and the beads were washed with 15. mu.L of protein extraction buffer (50 mM Tris. Cl pH 7.5; 150 mM NaCl; 1mM EDTA pH 8.0; 0.1% v/v Nonidet P-40; 1% v/v Triton X-100; 1mM PMSF; 1 xcOComplete Protease Inhibitor cocktail, Roche).
（5）PALBinding protein of promoter DNA fishing and separation: resuspending the protein containing the biotin labelPALThe magnetic beads of the promoter DNA are added into the rice Lemont natural total protein solution and then placed in a rotary mixer to be incubated for 4 h at 4 ℃. After the incubation is finished, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2min, storing the centrifugal tube in the magnetic frame, and carefully sucking the supernatant; mu.L of protein extraction buffer (50 mM Tris. Cl pH 7.5; 150 mM NaCl; 1mM EDTA pH 8.0; 0.1% v/v Nonidet P-40; 1% v/v Triton X-100; 1mM PMSF; 1 xcOComplete Protease Inhibitor cocktail, Roche) washed the beads 2 times, which keeps the centrifuge tubes in the magnetic rack. After washing, taking out the centrifuge tube, adding 15 mu L of 2 xSDS buffer for resuspending magnetic beads, boiling for 8 min at 100 ℃, taking out, standing to room temperature, placing in a magnetic frame, standing for 2min, slowly sucking out supernatant fluid to obtain a solution of promoter DNA binding protein, and separating the protein by adopting 10% v/v polyacrylamide gel electrophoresis. The results are shown in FIG. 3, in which the arrow in lane 3 is that of rice PI312777PALThe binding protein of the gene promoter, lane 4 is the negative control of the combination of the natural total protein of the leaf of the rice PI312777 and the streptavidin coupled magnetic beads, and the result shows that the protein contains rice LemontPALThe magnetic beads of the gene promoter can effectively fish the protein bound to the promoter DNA.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> method for effectively fishing rice promoter DNA binding protein
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence
<400> 1
ggtagtgcta gatatgaagg gctgc 25
<210> 2
<211> 25
<212> DNA
<213> Artificial sequence
<400> 2
gcacgaggag aagagaggat tcgat 25
Claims (1)
1. A method for effectively fishing a rice promoter DNA binding protein is characterized by comprising the following steps:
(1) extracting natural total protein of rice;
(2) amplifying promoter DNA and marking biotin;
(3) incubating promoter DNA marked by biotin and streptavidin coupled magnetic beads;
(4) fishing and separating the binding protein of promoter DNA;
the specific steps of the step (1) of extracting the natural total protein of the rice are as follows: putting 3-5 g of fresh rice leaves into a precooled mortar, and grinding the fresh rice leaves into powder under the condition of liquid nitrogen; quickly transferring the powder into a pre-cooled 50 ml centrifuge tube, adding a protein extraction buffer solution with the volume of 1.5-2 times that of tissue powder, fully and uniformly mixing by vortex oscillation, carrying out ice bath for 15-30 min, carrying out rotary incubation for 45-60 min at 4 ℃, centrifuging for 15-20 min at 15000g at 4 ℃, taking the supernatant, filtering residues in the supernatant by using sterilized Miracloth, further centrifuging the filtered supernatant for 15-20 min at 15000g at 4 ℃, and filtering a small amount of residues by using sterilized Miracloth again to finally obtain a rice natural total protein solution, and storing for later use at 4 ℃;
the specific method for amplifying the promoter DNA and labeling the biotin in the step (2) comprises the following steps: designing forward and reverse amplification primers aiming at a researched target promoter, wherein biotin (biotin) labeling is carried out on the 5' end of the forward primer, rice DNA is used as a template for amplification to obtain promoter DNA with the biotin label, and the promoter DNA is recovered and purified by agarose gel electrophoresis and stored at-20 ℃ for later use;
the target promoter is a PAL promoter, and the forward primer sequence is as follows:
5 '-GGTAGTGCTAGATATGAAGGGCTGC-3 '，
the reverse primer sequence is 5'-GCACGAGGAGAAGAGAGGATTCGAT-3';
the specific operation steps of the biotin-labeled promoter DNA and streptavidin coupled magnetic bead incubation in the step (3) are as follows: taking 15-20 mu L of streptavidin coupled magnetic beads to a 1.5 ml sterile centrifuge tube, placing the centrifuge tube in a magnetic frame, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking off a supernatant; taking out the centrifuge tube, adding 60-80 mu L Binding solution to lightly resuspend the magnetic beads, putting the centrifuge tube in a magnetic frame again after resuspension, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking off the supernatant; taking out the centrifuge tube, adding 60-80 mu L of Binding solution to lightly resuspend the magnetic beads, adding the biotin labeled promoter DNA with the same volume and the total volume of 10-15 pmole, lightly mixing, placing in a rotary mixer, and incubating for 3-4 h at room temperature; after incubation, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2-3 min, storing the centrifugal tube in the magnetic frame, and carefully sucking off a supernatant; taking out the centrifuge tube, adding 120-160 mu L of Washing solution for gently resuspending the magnetic beads, putting the centrifuge tube in a magnetic frame again after resuspension, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking off the supernatant; taking out the centrifuge tube, adding 120-160 mu L of sterile double distilled water to lightly resuspend the magnetic beads, putting the centrifuge tube into the magnetic frame again after resuspension, standing for 2-3 min, storing the centrifuge tube in the magnetic frame, and carefully sucking the supernatant; taking out the centrifuge tube, and resuspending the magnetic beads by using 15-20 mu L of protein extraction buffer solution; wherein the Binding solution and Washing solution are selected from Dynabeads kinase B DNA Kit of Saimer Feishale technologies, Inc.;
the specific operation steps of fishing and separating the binding protein of the promoter DNA in the step (4) are as follows: adding the magnetic beads containing the biotin-labeled promoter DNA re-suspended in the step (3) into the natural total protein solution of the rice obtained in the step (1), placing the mixture into a rotary mixer, and incubating the mixture for 3-4 hours at 4 ℃; after incubation, taking out the centrifugal tube, placing the centrifugal tube in a magnetic frame, standing for 2-3 min, storing the centrifugal tube in the magnetic frame, and carefully sucking off a supernatant; washing the magnetic beads for 2 times by using 120-160 mu L of protein extraction buffer solution, and keeping the centrifugal tube in a magnetic frame in the process; after washing, taking out the centrifuge tube, adding 15-20 mu L of 2 xSDS buffer for resuspending magnetic beads, cooking at 100 ℃ for 8-10 min, taking out, standing to room temperature, placing in a magnetic frame, standing for 2-3 min, slowly sucking out supernatant to obtain a promoter DNA binding protein solution, and separating protein by adopting 10% v/v polyacrylamide gel electrophoresis;
the 2 x SDS buffer contained the following final concentrations of components: 20% v/v glycerol, 0.02% w/v bromophenol blue, 4% w/v SDS, 0.2M Tris.Cl pH 6.8, 200 mM DTT;
the protein extraction buffer contained the following final concentrations of components: 50 mM Tris. Cl pH 7.5; 150 mM NaCl; 1mM EDTA pH 8.0; 0.1% v/v Nonidet P-40; 1% v/v Triton X-100; 1mM PMSF; 1 × cOmplete Protease Inhibitor cocktail.
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| US5484701A (en) * | 1990-01-26 | 1996-01-16 | E. I. Du Pont De Nemours And Company | Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products |
| CN104862384A (en) * | 2015-03-04 | 2015-08-26 | 福建师范大学 | Method for screening protein capable of interacting with promoter |
| CN107098948A (en) * | 2017-05-05 | 2017-08-29 | 南京农业大学 | Method of protein in a kind of rapid extraction fruit plum blossom bud |
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