CN103146685A - Chromatin co-immunoprecipitation technology by using Flag protein label precipitation target proteins - Google Patents
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Abstract
The invention discloses a chromatin co-immunoprecipitation technology by using Flag protein label precipitation target proteins. The chromatin co-immunoprecipitation technology is that a small section of Flag protein label sequence is added on a sequence of the target proteins; and an eukaryotic expression vector is transfected into a cell to express after the eukaryotic expression vector is built. In the process of carrying out chromatin co-immunoprecipitation, the traditional method is broken through, the immunoprecipitation is carried out by using the Flag protein label antibody instead of protein antibody. Compared with the traditional method, the method has the advantages that the specificity of the antibody precipitation target protein is improved, the application of the antibody is reduced, and the expense of experiment is greatly reduced.
Description
Technical field
The present invention is applied to chromatin Immunoprecipitation (the Chromatin Immunoprecipitation of a kind of researching DNA and protein interaction, ChIP), relate to and build on target protein with the expression vector of Flag albumen label and precipitate the method for DNA-albumen composition with its antibody.
Background technology
Chromatin Immunoprecipitation (ChIP) is a kind of method of studying in vivo transcription factor (DNA) and the regional direct interaction of target gene promoters, can directly determine in vivo the dynamic change of their interaction modes, can access the information of transcription factor binding site point, determine its direct target gene.Technique in early days is used to study the aspects such as the interaction of DNA and histone on nucleosome and histone modification more.In recent years, chromatin Immunoprecipitation (ChIP) has obtained many Improvement and perfections, becomes a kind of interactional widely used technology of specific DNA sequence on the interior transcriptional regulator of body and target gene promoters that is used to study.The ChIP technology has become the effective means the most of research gene expression regulation on the chromatin level.It is worth mentioning that, technique combines with DNA chip and molecule clone technology, can be used for the distribution situation of target gene on full genome that the known target protein of high-flux sequence screening mutually combines with it.This will help to seek out more transcription factor, for the pathogenesis of study of disease is verified road.
The principle of chromatin Immunoprecipitation (ChIP) is: under physiological status intracellular DNA and protein cross together, process after chromatin chip section by ultrasonic or enzyme, utilize the specific recognition reaction of Ag-Ab, the DNA fragmentation that will combine with target protein precipitates, there is the DNA fragmentation of histone modification or transcriptional control with enrichment, then detects the DNA sequence dna of this enrichment fragment by multiple detected downstream technology (quantitative PCR, gene chip, high-flux sequence etc.).
The ability of the DNA fragmentation of isolated protein combination from unconjugated fragment background is finally depended in the sensitivity of chromatin Immunoprecipitation (ChIP), and wherein the quality of antibody and IP step are crucial.Yet the non-specific binding of protein and genomic dna can produce different types of background, and these backgrounds can be crosslinked catches and is not subjected to that the IP step is improved to be affected.Because the nonspecific binding site of shorter dna fragment is less, so the non-specific binding level can reduce along with the minimizing of DNA fragmentation length.
Summary of the invention
The technical problem to be solved in the present invention is of poor quality for the antibody that occurs in the chromatin co-immunoprecipitation and the low problem of specificity that cause and a kind of brand-new method that proposes.
Present method is at first to need to build a target protein gene with the carrier for expression of eukaryon of Flag albumen sequence label, and whether the expression vector that detection successfully constructs is at cell inner expression; Secondly the expression vector transfection that builds is advanced in specific cell; At last with the cell formaldehyde crosslinking, the mixture of target protein and downstream target gene is removed to precipitate in ultrasonication with Flag albumen tag antibody (but not target protein antibody), and follow-up other steps are with traditional chromatin Immunoprecipitation.Specifically, present method comprises the steps:
1. carrier construction: find out the gene order of target protein on NCBI, pcr amplification goes out the purpose fragment, and the purpose fragment that amplifies is cloned into the carrier for expression of eukaryon of an end with the Flag sequence.
2. whether whether the carrier that detect to build expresses: extract plasmid, its transient transfection was advanced in specific cells 48 hours, carry total protein and be Western Blot and detect and express.
3. the expression vector transfection that builds is advanced in specific cells: can express if step 2 proves the expression vector that builds, then carry out this step.According to the difference of different cell type and expression amount, select transient transfection or stable transfection, the expression vector transfection that builds is advanced in specific cells, after reaching the expectation degree, the cell inner expression amount carry out step 4 until it again.
4. formaldehyde crosslinking cell: this step with traditional chromatin Immunoprecipitation (ChIP), is mainly with formaldehyde, intracellular protein-DNA, protein-protein to be fixed up.
5. ultrasonication: this step with traditional chromatin Immunoprecipitation (ChIP), is mainly with Ultrasonic Cell Disruptor, genomic dna in Protein-DNA complex to be smashed in certain length range, is generally 200bp-1kb.
6. remove to precipitate the mixture of target protein and downstream target gene with Flag albumen tag antibody (but not target protein antibody): this step is the core procedure of this method.Traditional chromatin Immunoprecipitation (ChIP) is to remove to precipitate the mixture of target protein and downstream target gene with target protein antibody, its existing greatest problem is that the efficient of antibody precipitation is low and the antibody usage quantity is large, and antibody is very expensive, yet this method is but to replace target protein antibody to remove to precipitate target protein-downstream target gene mixture with Flag albumen tag antibody.Because Flag albumen tag antibody specificity is very good, and antibody is expensive, so present method has solved low and the large and expensive problem of antibody usage quantity of antibody efficient that traditional chromatin Immunoprecipitation (ChIP) faces, has greatly saved expense.
7. subsequent operations step is all with traditional chromatin Immunoprecipitation (ChIP).
Present method has following advantage and effect:
1. at first present method is to have built a mistake expression system to do the chromatin co-immunoprecipitation, because improved target protein at intracellular expression amount, so can be used for studying the target gene site, downstream that in cell, low target protein of expressing regulates and controls.
2. present method replaces target protein antibody to do precipitation with Flag albumen tag antibody, has improved on the one hand the specificity of antibody precipitation, has reduced on the other hand the usage quantity of expensive antibody, has greatly saved expense.
3. present method is with the crossing in the expression system transfered cell of target protein, it to be expressed one at cell inner stablity, so lessly be subject to cell state and the low impact of oneself expression amount.
Description of drawings
The expression vector of Fig. 1 for building is specially N2ICD-3 * Flag-pcDNA3.0 plasmid, and the left side is the DNA Ladder of 1kb, and molecular weight is 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 8kb, 10kb successively, and target protein sequence N2ICD is 2.3kb.
Fig. 2 is that the expression vector that builds is in intracellular expression, be specially N2ICD-3 * Flag-pcDNA3.0 plasmid in the intracellular expression of BxPC3, target protein N2ICD size is 110kD, the left side for Flag antibody as primary antibodie, the right for Notch-2 antibody as primary antibodie.
Fig. 3 is the genomic fragment of ultrasonication, be specially genome is smashed in the 200-1kb scope, the left side be the DNA Ladder of 100bp, molecular weight is 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 1kb, 1.5kb successively.
Embodiment
Embodiment 1
We select born of the same parents' inner segment N2ICD of Notch-2 gene as target protein, study the target gene in its downstream, and its length is 2.3kb.Therefore, need to build one section N2ICD with the carrier for expression of eukaryon of Flag albumen sequence label (sequence of specific design is 3 * Flag sequence, can with reference to Sigma company), called after N2ICD-3 * Flag-pcDNA3.0.We select pancreatic cancer cell BxPC3 cell to do the experiment of chromatin co-immunoprecipitation.
The concrete steps of carrier construction can be with reference to " molecular cloning " third edition, and Science Press publishes.
1. amplify born of the same parents' inner segment N2ICD sequence of Notch-2 gene from cDNA library, its clone's forward (FWD) is had in the pcDNA3.0 plasmid of 3 * Flag sequence, whether pcr amplification detects and successfully N2ICD is cloned successfully, and as shown in Figure 1, the plasmid amplification that successfully constructs goes out purpose fragment 2.3kb.
2. whether detect N2ICD-3 * Flag-pcDNA3.0 plasmid expresses.The bacterium liquid of N2ICD-3 * Flag-pcDNA3.0 plasmid is advanced in the conversion of spending night and cultivate 10ml with aseptic glass test tube 37, second day upgrading grain, and plasmid concentration preferably can reach 500ng/ul, is beneficial to next step transfection.Transfection reagent specification sheets with reference to Roche company advances N2ICD-3 * Flag-pcDNA3.0 plasmid transient transfection in pancreatic cancer cell BxPC3, extracts total protein after 48 hours and is Western Blot and observes expression, the results are shown in Figure 2.
Following steps are applied to chromatin Immunoprecipitation (ChIP):
3. the carrier that determine to build advances N2ICD-3 * Flag-pcDNA3.0 plasmid transient transfection in pancreatic cancer cell BxPC3 referring again to the transfection reagent specification sheets of Roche company after the BxPC3 cell inner expression, expresses until it in 48 hours to begin to process cell after stable.
4. with formaldehyde, that cell is crosslinked fixing, crosslinked with the glycine termination, after lysing cell extracts nucleus, then smash genome with Ultrasonic Cell Disruptor, control parameter genome is smashed as shown in Figure 3 between 200bp-1kb.
5. remove the nonspecific proteins in system with Protein G albumen pearl (company's purchase), adding Flag albumen tag antibody carries out immunoprecipitation (note: this step is the core of present method again, to have replaced Notch-2 antibody with Flag albumen tag antibody), with Protein G albumen pearl, the target protein of Flag albumen tag antibody and connection thereof-DNA mixture is dragged down again, clean at last Protein G albumen pearl, separate Protein G albumen pearl and target protein-DNA mixture, 65 degree are separated crosslinked, released dna.
6. attached: traditional chromatin Immunoprecipitation (ChIP) is sought the downstream regulatory site of target protein N2ICD.
As follows:
Chromatin co-immunoprecipitation experiment (ChIP)
1.37 in the degree incubator, culture density is every dish 3 * 10
7Individual BxPC3 cell, serum starvation 48h.
2. the sealing treatment of Protein G agarose ball: get the good Protein G agarose ball of 300 μ l swellings, of short durationly abandon supernatant after centrifugal, add the 1ml distilled water, 4 ℃ of rotation 3min wash away residual salt and ethanol on vortex mixer, repeat to wash 3 times.Add distilled water 900 μ l, 10mg/ml BSA100 μ l, 4 ℃ of rotation overnight incubation, standby on vortex mixer.
3. process cell after Preset Time point at once.Add 4% formaldehyde solution in substratum, making its final concentration is 1%, after mixing at room temperature standing crosslinked 10 minutes.Add the 1.25M glycine, making its final concentration is 0.125M again, at room temperature standing 5 minutes termination crosslinking reactions after mixing.Outwell reaction solution, with culture dish as on ice.
4. with appropriate 4 ℃ of 1 * PBS, every dish cell is washed 2 times, blotted washing lotion.Every dish adds 1.2ml4 ℃ of 1 * PBS afterwards, scrapes cell with cell, moves in 1.5ml EP pipe.4 ℃ of centrifugal 5min of 5000rpm abandon supernatant, obtain cell precipitation.
5. add in every tube cell precipitation 300 μ l4 ℃ Lysis buffer, final concentration be 1 * 25 * cocktail and final concentration be the PMSF of 1mM, blow and beat resuspended mixing, vortex vibration 3-5 time is at standing cracking 10min on ice.4 ℃ of centrifugal 5min of 8000rpm, abandon supernatant afterwards, obtains the nucleus precipitation.
6. add in every tube cell core precipitation 300ul4 ℃ of nucleus lysate, final concentration be 1 * 25 * cocktail and final concentration be the PMSF of 1mM, blow and beat resuspended mixing, vortex vibration 3-5 time is at standing cracking 10min on ice.
7. on ice ultrasonic 3 times, ultrasound intensity 50, each 5s, every minor tick 1min.4 ℃ of centrifugal 10min of 12000rpm, get supernatant afterwards.
8. every pipe equal-volume supernatant that takes a morsel is incorporated in 1 pipe as the Input sample, and cumulative volume 50 μ l get final product.Add 50 μ l distilled waters, final concentration is NaCl and the 2 μ l RNase A of 0.2M, hatches 30min for 37 ℃ after mixing again.Add afterwards 2 μ l Proteinase Ks, separate crosslinked spending the night for 65 ℃.The phenol/chloroform extracting, 1% agarose gel electrophoresis is identified ultrasonic clip size, is advisable with 200-500bp.
9. the homogeneous Protein G agarose ball suspension (total consumption 500 μ l) that adds equal-volume to seal in the ultrasonic supernatant liquor of every pipe, final concentration is 1 * 25 * cocktail, 4 ℃ of rotations are hatched and were carried out pre-washing in 2 hours on vortex mixer, with the nonspecific immunity sphaeroprotein in the system of removing.4 ℃ of centrifugal 2min of 5000rpm, collect supernatant afterwards.
Every pipe add final concentration be 1 * 25 * cocktail, again in wherein adding 5 μ g Normal Rabbit IgG non-specific antibodies as negative control in a pipe, add 5 μ g specific antibodies (comprising positive control antibody) in other every pipes, 4 ℃ of rotation overnight incubation on vortex mixer.
11. equivalent after second half Protein G agarose ball suspension mixing of remainder is added in each pipe, then add final concentration be 1 * 25 * cocktail, 4 ℃ of rotations are hatched and were carried out immunoprecipitation in 2 hours on vortex mixer.4 ℃ of centrifugal 2min of 5000rpm, abandon supernatant afterwards, obtains being combined with the Protein G agarose ball precipitation of DNA-transcription factor-transcription factor antibody complex.
12. under 4 ℃ of environment, on vortex mixer, Protein G agarose ball precipitation is respectively washed 1 time with 1ml low salt buffer, high-salt buffer and LiCl damping fluid respectively, use at last TE (pH8.0) damping fluid to wash twice, each 5min, 4 ℃ of centrifugal 2min of 5000rpm abandon supernatant.
13. prepare elutriant.Add 300 μ l elutriants resuspended in every tubulin G agarose ball precipitation, 65 ℃, 1200rpm vibration 30min carry out wash-out on the heating vortex mixer.The centrifugal 2min of normal temperature 5000rpm, collect supernatant afterwards.Adding final concentration in every pipe supernatant is NaCl and the 2 μ l Proteinase Ks of 0.2M, separates crosslinked spending the night for 65 ℃, and the phenol/chloroform extracting obtains the target DNA of purifying.
14.PCR (using HES-1 promotor specific sequence as primer) identified in amplification.Delivery plate, each 2 μ l of dNTP, 10 * LA Taq buffer, each 1 μ l of upstream and downstream primer, distilled water 12ul and Pfu high-fidelity DNA polymerase 0.2 μ l add in the little EP pipe of thin-walled, and fully mixing, carry out pcr amplification in the mode of warm start.Programming is that 94 ℃ of 5min denaturations, 94 ℃ of 30s sex change, annealing 30s, 72 ℃ are extended 30s, and amplification 30 is taken turns.
Claims (7)
1. this method is chromatin co-immunoprecipitation (the Chromatin Immunoprecipitation of a kind of Flag of utilization albumen label precipitation target protein and target gene, ChIP) technology, its main purpose is the research transcription factor, the unique distinction of this method is: build the expression vector that a target protein gene order is connected with Flag albumen sequence label, near transfection is advanced in specific cells, utilizes the chromatin Immunoprecipitation (ChIP) of the DNA-albumen composition of Flag albumen tag antibody precipitation target protein and target gene.
2. technological method according to claim 1, is characterized in that substituting target protein antibody with Flag albumen tag antibody carries out chromatin co-immunoprecipitation (ChIP), and its feature is: a. has improved the specificity of traditional C hIP; B. reduced the use of antibody.
3. technological method according to claim 2 is characterized in that comprising the following steps:
(1) build the carrier for expression of eukaryon (plasmid) that the target protein gene order is connected with 3 * Flag albumen sequence label;
(2) expression vector (plasmid) transfection that builds is advanced in specific clone to express;
(3) with the DNA-albumen composition of Flag albumen tag antibody precipitation target protein and its target gene, its subsequent operations is with traditional chromatin Immunoprecipitation (ChIP).
4. technological method according to claim 3 is characterized in that step (1) builds the carrier for expression of eukaryon (plasmid) that the target protein gene order is connected with 3 * Flag albumen sequence label and is:
Build a carrier for expression of eukaryon, its requirement: the N end of target protein or C end are connected with 3 * Flag albumen sequence label of a bit of 100bp of being no more than, after the vector expression that successfully constructs, the N of target protein end or C hold the Flag albumen label with a section very short, this Flag albumen label can be combined with Flag albumen tag antibody, and target protein is precipitated.
5. technological method according to claim 3 is characterized in that step (2) advances expression vector (plasmid) transfection that builds in specific clone to be expressed as:
The selected a kind of clone that will study is advanced carrier for expression of eukaryon transient transfection or the stable transfection that builds in cell to express.
6. technological method according to claim 3 is characterized in that step (3) with the DNA-albumen composition of Flag albumen tag antibody precipitation target protein and its target gene is:
With formaldehyde, that cell is crosslinked, carry out again thereafter the ultrasonic genome of smashing, then precipitate the DNA-albumen composition of goal gene and its target gene with Flag albumen tag antibody, with specific pearl, mixture is dragged down, separate at last the trace dna that crosslinked recovery obtains.
7. technological method according to claim 2, the characteristics of present method are:
A. improved the specificity of traditional C hIP: the specificity of being combined with the albumen label due to Flag albumen tag antibody is very good, so present method is high than the specificity of traditional C hIP, can reduce non-specific background, improves confidence level;
B. reduced the use of antibody: traditional method needs a large amount of target protein antibody to improve specificity, and this method can reduce the use of antibody, has saved certain expense.
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| CN105463090A (en) * | 2015-12-21 | 2016-04-06 | 同济大学 | Index-first chromatin immunoprecipitation (iChIP) high-throughput sequencing experimental method applied to zebrafish embryos |
| CN106596974A (en) * | 2016-12-16 | 2017-04-26 | 中南大学 | Inflammatory myopathy SAE1 self antibody non-radioactive detection method and application thereof |
| CN112941159A (en) * | 2020-12-25 | 2021-06-11 | 南京农业大学 | Method for identifying guanine quadruplet locus of plant genome DNA at whole genome level |
| CN113046381A (en) * | 2021-04-12 | 2021-06-29 | 南华大学 | Method for separating specific protein-DNA complex in organism, fusion protein and preparation method thereof |
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