CN103290091A - Protein interaction detection method with low false positive rate - Google Patents
Protein interaction detection method with low false positive rate Download PDFInfo
- Publication number
- CN103290091A CN103290091A CN2012100775294A CN201210077529A CN103290091A CN 103290091 A CN103290091 A CN 103290091A CN 2012100775294 A CN2012100775294 A CN 2012100775294A CN 201210077529 A CN201210077529 A CN 201210077529A CN 103290091 A CN103290091 A CN 103290091A
- Authority
- CN
- China
- Prior art keywords
- protein
- fluorescin
- false positive
- detection method
- positive rate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a protein interaction detection method with low false positive rate, relates to a detection system and belongs to the field of a molecular biological technology. According to the method, two generally needed bimolecular fluorescence complementation (BiFC) detection systems based on single expression plasmid vectors are built in a dual expression plasmid vector system by a BiFC technology based on fluorescent protein, so the false positive rate of the BiFC detection method in research on protein-protein interaction can be reduced obviously, and quantitative analysis can be realized. The method also can be applied to screening research on unknown protein-protein interaction based on a gene library and detection on protein-protein interaction in living animals.
Description
Technical field
The invention belongs to technical field of molecular biology, particularly a kind of protein interaction detection method of low false positive rate.
Background technology
Cell itself is the elementary cell that is formed an orderly vital movement by the different biological molecules interphase interaction.The generation of the great vital movement of cell and adjusting interact by (protein-protein, protein-nucleic acid etc.) between biomacromolecule and realize.Protein-protein interaction (protein-protein interaction, hereinafter to be referred as " PPI ") not only controlling genetic transcription, cell fission and cell proliferation, go back signal transduction, oncogenic transformation and adjustment etc. in the mediated cell vital movement process simultaneously, therefore studying protein-protein interaction has important life science meaning.
At present, can realize the technology of accurate sterically defined PPI imaging in the viable cell at mammalian cell, has only FRET (fluorescence resonance energy transfer) (Fluorescence Resonance Energy Transfer, FRET) technology and bimolecular fluorescence complementary (Bimolecular Fluorescence Complementation, BiFC) technology.BiFC technology based on fluorescin, refer to utilize gene clone technology that complete fluorescin is cut in appropriate site and form two non-blooming fragments, and make these two fragments constitute fusion rotein with two target proteins respectively, by the further space length of these two fluorescence light segments of the interaction between two target proteins, thus the fusion rotein that is folded to form structural integrity again and has fluorescent characteristic.Compare with the FRET technology, bimolecular fluorescence complementary technology is lower to the performance requriements of plant and instrument, experimental result is more directly perceived, subsequent disposal is simple, detection sensitivity is higher, especially have advantage in the detection of weak PPI.But existing BiFC technology all is accompanied by higher false positive rate, also can produce very high background signal in detection, these shortcomings make the interaction of this method between agnoprotein matter detect and the quantitative examination of PPI in be restricted.Therefore, the BiFC system of the low false positive rate of development will greatly promote intravital protein function research.In addition, utilize the BiFC technology based on multiple different fluorescins can realize many detections to protein interaction in the viable cell easily.Usually, when carrying out the BiFC detection at a pair of PPI, need dye two kinds of plasmids to transit cell simultaneously.Detect if carry out BiFC at three couples of PPI simultaneously, then need to dye 6 kinds of plasmids to transit cell simultaneously.In obvious many plasmid transfections process, heterogeneic expression ratio is difficult to control.This problem has influenced the BiFC technology to a great extent in many application during protein interaction is detected.
Summary of the invention
The objective of the invention is provides a kind of protein interaction of low false positive rate detection method for addressing the above problem.Utilize this method when detecting protein interaction, the false positive that can reduce BiFC significantly produces.
The technical solution adopted in the present invention is:
A kind of protein interaction detection method of low false positive rate may further comprise the steps:
1) fluorescin is cut into two protein sequence fragments at specific site, the N end of fluorescin and C end be called after FPN and FPC respectively;
2) downstream that dna sequence dna and the FPN gene order of target protein A to be measured is inserted into a promotor in the dual-expression vector simultaneously, another testing protein B and insert another promotor downstream in the dual-expression vector simultaneously with the FPC gene order, thus made up the bimolecular fluorescence complementary system based on dual-expression vector;
3) will contain the bimolecular fluorescence complementary system transfectional cell of target protein A to be measured and B simultaneously, cultivate after 16～24 hours, use fluorescence microscope source stimulating front and back whether to have fluorescence; Interact if two target proteins exist, then the N of fluorescin end and C end are close, thus spontaneous complete fluorescin, the generation fluorescent signal of being reassembled as; If there is not or has only extremely faint fluorescence, prove not have between two target proteins to interact.
Preferably, described fluorescin is DsRed mLumin far away, yellow fluorescence protein mVenus and cyan fluorescent protein mCerulean.
Preferably, described dual-expression vector is pBudCE4.1, pIRES2 and pVITR03.
Preferably, in the described step 3), interact when two target proteins exist, the N end of fluorescin and the close distance range of C end are 10nm～20nm, thus spontaneous complete fluorescin, the generation fluorescent signal of being reassembled as.
Further, when described fluorescin was DsRed mLumin far away, specific site was No. 155 Serine.
Further, when described fluorescin was yellow fluorescence protein mVenus, specific site was No. 155 Serines and No. 173 Serines.
Further, when described fluorescin was cyan fluorescent protein mCerulean, specific site was No. 155 Serines and No. 173 Serines.
The present invention has the following advantages:
Present method can satisfy under the viable cell condition and detects protein interactions, dual-expression vector system based on fluorescin, compare with BiFC system (the common transfections of at least two carrier molecules) commonly used at present, operate easier, the carrier amount of transfection is more easy to control, every pair of Expression of Fusion Protein amount is more stable, can be used for the quantitative analysis of protein interaction; This method has significantly reduced the false-positive generation of BiFC simultaneously, has enlarged BiFC The Application of Technology scope, makes it to be applicable to filter out unknown protein-protein interaction quickly and efficiently from gene library.
Description of drawings
The present invention is further detailed explanation below in conjunction with drawings and embodiments.
Fig. 1 is the dual-expression vector plasmid map of mLumin-BiFC system;
Fig. 2 distinguishes transfection pBud-Ln-bFos-Lc-bJun, pBud-Ln-b Δ Fos-Lc-bJun, pBud-Ln-bFos-bJun-Lc and pBud-Ln-b Δ Fos-bJun-Lc in 37 ℃ of cultivation fluorescence imaging figure after 24 hours for the COS-7 cell;
Fig. 3 distinguishes transfection pBud-Ln-bFos-Lc-bJun, pBud-Ln-b Δ Fos-Lc-bJun, pBud-Ln-bFos-bJun-Lc and pBud-Ln-b Δ Fos-bJun-Lc in the data statistic analysis histogram of 37 ℃ of cultivations after 24 hours for the COS-7 cell;
Fig. 4 for the COS-7 cell respectively transfection pBud, pBud-Ln-b Δ Fos-bJun-Lc and pBud-Ln-bFos-bJun-Lc in 37 ℃ of flow cytometry analysis after cultivating 24 hours figure as a result;
Fig. 5 for the COS-7 cell respectively transfection pBud, pBud-Ln-b Δ Fos-bJun-Lc and pBud-Ln-bFos-bJun-Lc in 37 ℃ of data statistic analysis histograms after cultivating 24 hours;
Fig. 6 distinguishes transfection bJun-Vn/bFos-Vc, bJun-Vn/ Δ bFos, pBud-Vn-bFos-Vc-bJun and pBud-Vn-b Δ Fos-Vc-bJun in 37 ℃ of cultivation fluorescence imaging figure after 24 hours for the COS-7 cell;
Fig. 7 distinguishes transfection bJun-Vn/bFos-Vc, bJun-Vn/ Δ bFos, pBud-Vn-bFos-Vc-bJun and pBud-Vn-b Δ Fos-Vc-bJun in the data statistic analysis histogram of 37 ℃ of cultivations after 24 hours for the COS-7 cell;
Fig. 8 distinguishes transfection pBud-Ln-RBD-Lc-bKRas, pBud-Ln-RBD-Lc-KRas12v, pBud-Ln-RBD-Lc-KRas185S in 37 ℃ of cultivation fluorescence imaging figure after 24 hours for the COS-7 cell;
Fig. 9 distinguishes transfection pBud-Ln-RBD-Lc-bKRas, pBud-Ln-RBD-Lc-KRas12v, pBud-Ln-RBD-Lc-KRas185S in the data statistic analysis histogram of 37 ℃ of cultivations after 24 hours for the COS-7 cell;
Figure 10 distinguishes transfection pBud-Ln-RBD-Lc-bKRas, pBud-Ln-RBD-Lc-KRas12v, pBud-Ln-RBD-Lc-KRas185S in the laser co-focusing fluorescence imaging figure of the individual cells of 37 ℃ of cultivations after 24 hours for the COS-7 cell;
Figure 11 distinguishes transfection pBud-Ln-Grb2-Lc-bKRas, pBud-Ln-Grb2-Lc-KRas12v, pBud-Ln-Grb2-Lc-KRas185S in 37 ℃ of cultivation fluorescence imaging figure after 24 hours for the COS-7 cell;
Figure 12 distinguishes transfection pBud-Ln-Grb2-Lc-bKRas, pBud-Ln-Grb2-Lc-KRas12v, pBud-Ln-Grb2-Lc-KRas185S in the data statistic analysis histogram of 37 ℃ of cultivations after 24 hours for the COS-7 cell;
Figure 13 distinguishes transfection pBud-Ln-Grb2-Lc-bKRas, pBud-Ln-Grb2-Lc-KRas12v, pBud-Ln-Grb2-Lc-KRas185S in the laser co-focusing fluorescence imaging figure of the individual cells of 37 ℃ of cultivations after 24 hours for the COS-7 cell.
Embodiment
The present invention is a kind of detection method of protein interaction, and based on red fluorescent protein mutant far away and yellow fluorescence protein, and the key of this detection method is, choosing of fluorescin and choosing of mammalian cell dual-expression vector.That select for use in this embodiment is DsRed mLumin far away and yellow fluorescence protein mVenus, and dual-expression vector uses carrier for expression of eukaryon pBudCE4.1.PBudCE4.1 in the embodiment contains two strong promoter CMV and EF-1 α, insert N-terminal and the bFos of far red/yellow fluorescence protein fragment respectively in the downstream of CMV promotor, C-terminal and the bJun of far red/yellow fluorescence protein fragment inserted in EF-1 α promotor downstream.Make that fluorescin fragment and bFos/bJun can amalgamation and expressions, two fluorescin fragment complementations of mediation form a complete fluorescin that can be luminous under the interaction of bFos and bJun.In principle, also can substitute these two promotors with other strong promoter, but should guarantee that two promotors ability that promotor gene is expressed in identical carrier is suitable.
Fig. 1 is the dual-expression vector plasmid map of mLumin-BiFC system.Two fragments (mLumin-155C and mLumin-155N) that DsRed mLumin the 155th amino acids far away is divided into are by the complementation of PPI mediation fluorescin, formation can be luminous complete fluorescin mLumin, thereby the PPI optical imagery that can realize the mLumin-BiFC probe detects.When making up plasmid, used pBudCE4.1 dual-expression vector contains two of CMV and EF-1 α independently promotor and Zeocin screening resistance.By restriction endonuclease Sal I/BamH I and NotI/Bgl II fluorescence light segments mLumin-155N-bFos and mLumin-155C-bJun are inserted into CMV and EF-1 α promotor downstream respectively, make its can be simultaneously expressed fusion protein independently.Can be via the interaction between bFos and the bJun under 37 ℃ of conditions, make the fluorescin fragment mutually near and form complete fluorescin that can be luminous, produce fluorescent signal.Other probe involved in the present invention also can use other restriction enzyme site in the pBud CE4.1 carrier multiple clone site to carry out similar molecular cloning operation, also can replace bFos/bJun, be configured to the similar mLumin-BiFC probe for detection of other PPI.
Fig. 2 and Fig. 3 are the fluorogram of DsRed mutant mLumin-BiFC system far away behind transfection COS-7 cell based on dual-expression vector.As can be seen from Figure 2, in this individual system, the cell number of express fluorescent protein has very big difference in the positive and negative group.Almost can't see the cell that sends red fluorescence in negative control group, Fig. 3 illustrates that also the mLumin-BiFC system based on dual-expression vector has produced difference significantly in positive and negative group, reduced false-positive generation.
Fig. 4 and Fig. 5 are the flow cytometry of DsRed mutant mLumin-BiFC system far away behind transfection COS-7 cell based on dual-expression vector.The result shows that the mLumin-BiFC fluorescent signal positive based on dual-expression vector can reach 37.65 times with the feminine gender ratio.And the difference between negative group and the blank group is also very little.This explanation can reduce false positive results greatly based on the mLumin-BiFC system of dual-expression vector, and positive group has very strong fluorescence complementary efficient simultaneously.
Fig. 6 to Fig. 7 is for being building up to (Vn-bFos/ Δ bFos+Vc-bJun) in two expression vectors respectively and being implemented in the plasmid of (Vn-bFos/ Δ bFos-Vc-bJun) in the dual-expression vector, the fluorogram behind the transfection COS-7 cell based on the BiFC system of yellow fluorescence protein mutant mVenus.As can be seen from the figure, at present the most frequently usedly be implemented in two BiFC systems in the strongly expressed carrier, negative control still has a large amount of cells to produce fluorescent signals.And in dual-expression vector, the cell quantity and the fluorescence intensity that produce fluorescent signal in the negative control group all significantly decrease.
Previous work of the present invention is the structure of BiFC intermediate carrier.
Its concrete steps are:
BiFC intermediate carrier pBud-Vn-Vc and pBud-Ln-Lc plasmid construction
After cutting by the pcr amplification enzyme, mlumin encoding sequence 1-155 (Ln), 156-234 (Lc) insert Hind III/Sal I site and the Bgl II/Mlu I site of pBudCE4.1 respectively;
The used primer sequence of building process is
Embodiment 1
This embodiment is to utilize the detection method of setting up a kind of protein interaction of low false positive rate based on the mLumin-BiFC system of dual-expression vector.Based on this purpose, bFos is linked to each other (hereinafter to be referred as LN) with the N end of mLumin protein sequence, bJun holds link to each other (hereinafter to be referred as LC) with the C of mLumin protein sequence.
With reference to Fig. 1, Fig. 1 is based on the BiFC molecular probe structure iron in mLumin155 site, organizes mLumin-Ln55-Δ bFos/mLumin-Lc155-bJun by positive group mLumin-Ln155-bFos/mLumin-Lc155-bJun and the feminine gender of probe and constitutes.Wherein Δ bFos is the bFos after the deletion mutantion, can not interact with bJun under the physiological condition, thereby not produce the BiFC fluorescent signal.
Its concrete steps are: at first using gene engineering technique makes up pBud-Ln-Lc, and plasmid can be expressed Ln and Lc respectively in mammalian cell.Use increase respectively bFos (or Δ bFos) and clone of round pcr and insert Ln downstream among the pBud-Ln-Lc; (bJun and bFos can interact for the Lc upstream of amplification bJun and clone's insertion pBud-Ln-Lc or downstream, bJun and Δ bFos can not interact), namely make up four groups of plasmid vectors, be respectively pBud-Ln-Fos-Lc-Jun, pBud-Ln-Fos-Jun-Lc, pBud-Ln-Δ Fos-Lc-Jun and pBud-Ln-Δ Fos-Jun-Lc, thereby obtain to express the plasmid of BiFC molecular probe.Namely finishing BiFC probe involved in the present invention makes up.
Fig. 2 and Fig. 3 are based on dual-expression vector BiFC probe fluorogram and BiFC efficient statistics in the PPI detection application in cell in mLumin155 site.As scheme to show, respectively with behind pBud-Ln-Fos-Lc-Jun and the pBud-Ln-Fos-Jun-Lc transfection COS-7 cell, (spectral filter parameter 520-550 under the common fluorescent microscope, DM565,580LP, Olympus), observe and find that very strong red fluorescence signal is arranged, and pBud-Ln-Δ bFos-Lc-Jun and pBud-Ln-Δ bFos-Jun-Lc probe almost do not detect the red fluorescence signal.This explanation has tangible yin and yang attribute contrast gradient based on the mLumin-BiFC probe of dual-expression vector, proves that namely the present invention can reduce false-positive generation, can be applied to the research of viable cell internal protein-protein interaction more easily.
Fig. 4 and Fig. 5 are based on the fluidic cell detected result of the mLumin-BiFC system of dual-expression vector.For further checking the present invention can reduce the generation of false positive results, detected in the moon/positive test group far away red fluorescent signal after 10000 cell fluorescence complementations respectively by flow cytometry.Contrast the fluorescent signal per-cent of negative experimental group and blank group, more definite the present invention can be lowered the fluorescent signal of negative experimental group.Show that with the comparing result of positive experimental group the present invention can effectively reduce false positive and produce.
Fig. 6 and Fig. 7 are based on fluorogram and the statistics analysis behind the transfection COS-7 of the mVenus-BiFC system cell of dual-expression vector.As shown in the figure, by the BiFC system of two expression vectors of contrast use, find also can reduce false positive results based on the mVenus-BiFC system of dual-expression vector.
Embodiment 2
Raf1 is the main effects albumen of the important composition molecule KRas of cell signal approach, plays an important role in growth, growth and cancer take place.The Ras of Kras-Raf1 albumen is taken as the special probe of Ras activity in many researchs in conjunction with territory RBD.The KRas of GTP combining form has stronger avidity than KRas and the RBD of GDP combination, and the Ras of GDP combination and the avidity between Raf1 are because too weak and be not enough to participate in the signal conduction.Therefore, the allos dimerization of RBD and KRas can be used as the good protein interaction model of PPI detection method in the development viable cell.
Fig. 8 to Figure 10 is the fluorogram behind pBud-Ln-RBD-Lc-KRas, pBud-Ln-RBD-Lc-KRas 12v and the pBud-Ln-RBD-Lc-KrasC185S transfection COS-7 cell.In RBD and KRas or KRas mutant insertion pBud-Ln-Lc carrier, three groups of probes of pBud-Ln-RBD-Lc-KRas, pBud-Ln-RBD-Lc-KRas12v and pBud-Ln-RBD-Lc-KrasC185S have been made up.Behind the transfection COS-7 cell, under 37 ℃ of conditions, cultivate 24h respectively.Fluorescence microscope is found to have produced red fluorescence on cytolemma or in the full cell, shows that the interaction of the Kras of RBD and different structure occurs on the different spatial of cell.This result conforms to previous bibliographical information.And the red fluorescence signal that RBD and enhancement type KRas12v interaction produce obviously is better than natural type KRas.As seen genotype probe biomolecule provided by the present invention can detect protein interactions delicately in mammalian cell.
Embodiment 3
Grb2 is the important adaptin in the cell signal path, by Grb2, KRas can with different signal path downstream egg white hair looks mutual effects.The activation of KRas is mainly finished by itself and the effect of Grb2-SOS1 protein complexes.
Segment Grb2 and KRas or its mutant clone be will merge to pBud-Ln-Lc, pBud-Ln-Grb2-Lc-KRas, pBud-Ln-Grb2-Lc-KRas 12v and pBud-Ln-Grb2-Lc-KRas185S constructed.Behind the plasmid difference transfection COS-7 cell, after cultivating 24 hours under 37 ℃ of conditions, carry out imaging.With reference to Figure 11 to Figure 13, the red fluorescence of cell on cytolemma that pBud-Ln-Grb2-Lc-KRas12v expresses obviously is better than pBud-Ln-Grb2-Lc-KRas, in pBud-Ln-Grb2-Lc-KRas185S, then can't see tangible fluorescence, illustrate that the BiFC probe can be applied in the detection of the complex body that forms more than two albumen, the activation of also having verified KRas has strengthened the keying action between KRas and Grb2.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (7)
1. the protein interaction detection method of a low false positive rate is characterized in that, may further comprise the steps:
1) fluorescin is cut into two protein sequence fragments at specific site, the N end of fluorescin and C end be called after FPN and FPC respectively;
2) downstream that dna sequence dna and the FPN gene order of target protein A to be measured is inserted into a promotor in the dual-expression vector simultaneously, another testing protein B and insert another promotor downstream in the dual-expression vector simultaneously with the FPC gene order, thus made up the bimolecular fluorescence complementary system based on dual-expression vector;
3) will contain the bimolecular fluorescence complementary system transfectional cell of target protein A to be measured and B simultaneously, cultivate after 16～24 hours, use fluorescence microscope source stimulating front and back whether to have fluorescence; Interact if two target proteins exist, then the N of fluorescin end and C end are close, thus spontaneous complete fluorescin, the generation fluorescent signal of being reassembled as; If there is not or has only extremely faint fluorescence, prove not have between two target proteins to interact.
2. the protein interaction detection method of low false positive rate according to claim 1 is characterized in that, described fluorescin is DsRed mLumin far away, yellow fluorescence protein mVenus and cyan fluorescent protein mCerulean.
3. the protein interaction detection method of low false positive rate according to claim 1 is characterized in that, described dual-expression vector is pBudCE4.1, pIRES2 and pVITR03.
4. according to the protein interaction detection method of each described low false positive rate of claim 1 to 3, it is characterized in that, in the described step 3), when existing, two target proteins interact, the N end of fluorescin and the close distance range of C end are 10nm～20nm, thereby spontaneous complete fluorescin, the generation fluorescent signal of being reassembled as.
5. according to the protein interaction detection method of each described low false positive rate of claim 1 to 3, it is characterized in that when described fluorescin was DsRed mLumin far away, specific site was No. 155 Serine.
6. according to the protein interaction detection method of each described low false positive rate of claim 1 to 3, it is characterized in that when described fluorescin was yellow fluorescence protein mVenus, specific site was No. 155 Serines and No. 173 Serines.
7. according to the protein interaction detection method of each described low false positive rate of claim 1 to 3, it is characterized in that when described fluorescin was cyan fluorescent protein mCerulean, specific site was No. 155 Serines and No. 173 Serines.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210077529.4A CN103290091B (en) | 2012-03-22 | 2012-03-22 | A kind of Protein interaction detection method of low false positive rate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210077529.4A CN103290091B (en) | 2012-03-22 | 2012-03-22 | A kind of Protein interaction detection method of low false positive rate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103290091A true CN103290091A (en) | 2013-09-11 |
| CN103290091B CN103290091B (en) | 2015-07-29 |
Family
ID=49091619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210077529.4A Active CN103290091B (en) | 2012-03-22 | 2012-03-22 | A kind of Protein interaction detection method of low false positive rate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103290091B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226522A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of Cys C detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
| CN108226520A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
| CN108267435A (en) * | 2018-03-01 | 2018-07-10 | 北京大学 | Novel cell fibril framework labeling method based on bimolecular fluorescence complementary |
| CN108519484A (en) * | 2018-03-01 | 2018-09-11 | 北京大学 | The protein interaction in living cells is imaged and tracked using the bimolecular fluorescence complementary technology from connection label |
| CN108546709A (en) * | 2018-03-10 | 2018-09-18 | 东北农业大学 | The construction method of multi-functional plant binary plasmid pPBEL-BiFC and its application |
| CN114836470A (en) * | 2021-02-02 | 2022-08-02 | 中国科学技术大学 | Vector for expressing combined fluorescent protein fragment and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101620233A (en) * | 2009-05-27 | 2010-01-06 | 华中科技大学 | Method for detecting interaction of proteins |
-
2012
- 2012-03-22 CN CN201210077529.4A patent/CN103290091B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101620233A (en) * | 2009-05-27 | 2010-01-06 | 华中科技大学 | Method for detecting interaction of proteins |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226522A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of Cys C detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
| CN108226520A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
| CN108267435A (en) * | 2018-03-01 | 2018-07-10 | 北京大学 | Novel cell fibril framework labeling method based on bimolecular fluorescence complementary |
| CN108519484A (en) * | 2018-03-01 | 2018-09-11 | 北京大学 | The protein interaction in living cells is imaged and tracked using the bimolecular fluorescence complementary technology from connection label |
| CN108546709A (en) * | 2018-03-10 | 2018-09-18 | 东北农业大学 | The construction method of multi-functional plant binary plasmid pPBEL-BiFC and its application |
| CN114836470A (en) * | 2021-02-02 | 2022-08-02 | 中国科学技术大学 | Vector for expressing combined fluorescent protein fragment and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103290091B (en) | 2015-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shemesh et al. | Precision calcium imaging of dense neural populations via a cell-body-targeted calcium indicator | |
| Giladi et al. | Dissecting cellular crosstalk by sequencing physically interacting cells | |
| CN103290091B (en) | A kind of Protein interaction detection method of low false positive rate | |
| CN101620233B (en) | Method for detecting interaction of proteins | |
| Hurlock et al. | Identification of novel synaptonemal complex components in C. elegans | |
| Gao et al. | Multiplexed dendritic targeting of α calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein RNAs by the A2 pathway | |
| Plummer et al. | Expanding the power of recombinase-based labeling to uncover cellular diversity | |
| Paschke et al. | Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum | |
| Viktorinová et al. | The cadherin Fat2 is required for planar cell polarity in the Drosophila ovary | |
| CN106574292A (en) | Method for identifying desired cell type using miRNA expression as indicator | |
| Lossi et al. | Ex vivo imaging of active caspase 3 by a FRET-based molecular probe demonstrates the cellular dynamics and localization of the protease in cerebellar granule cells and its regulation by the apoptosis-inhibiting protein survivin | |
| Field et al. | Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs | |
| Zhang et al. | Fast super-resolution imaging technique and immediate early nanostructure capturing by a photoconvertible fluorescent protein | |
| Bhattacharyya et al. | Using Gjd3-CreEGFP mice to examine atrioventricular node morphology and composition | |
| Westbrook et al. | Collective signalling drives rapid jumping between cell states | |
| Christou-Kent et al. | CEBPA phase separation links transcriptional activity and 3D chromatin hubs | |
| CN104844691A (en) | Sox2 protein peptide aptamer and authenticating method thereof | |
| CN103145820B (en) | Cyclic-permutation fluorescent protein and preparation method and application of cyclic permutation fluorescent protein | |
| Larsen et al. | Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis | |
| CN106589061A (en) | Sox2-CDP binding domain candidate peptide aptamer | |
| CN113567402A (en) | Application of cAMP fluorescent probe G-flash 1 | |
| CN110095610A (en) | A kind of high throughput protein analysis method and its applicable library | |
| Wang et al. | Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion | |
| CN109897827B (en) | Construction method and application of zebra fish model for researching stress particles | |
| Hurlock | Identification and Characterization of Novel Synaptonemal Complex Components in C. elegans |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |