CN101105493B - Method for detecting protein interaction by immunological coprecipitation based on protein chip and reagent kit for detecting protein interaction - Google Patents
Method for detecting protein interaction by immunological coprecipitation based on protein chip and reagent kit for detecting protein interaction Download PDFInfo
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Abstract
Protein-protein interaction (PPI) is important for all biological processes; therefore, the research of protein interaction is always a research hot in the fields of cell biology and molecular biology. The co-immunoprecipitation (CoIP) is a classic method which is used to research protein interaction based on the specific action between antibody and antigen and is the commonest and most effectivemethod to find and testify PPI; the traditional CoIP steps based on a resin are complicated and labor consuming and are hard to realize the flux detection. The key point in the invention is that CoIPis implemented on glass aldehyde sheet of the fixed anti-label antibody by utilizing the epitope label of recombinant protein; protein-protein interaction (PPI) is detected by the antibody labeled byfluorescence. The method greatly simplifies the operation steps of the traditional CoIP, greatly improves the flux of sample detection and is a novel technique platform to research PPI simply, conveniently and swiftly and with high flux.
Description
Technical field:
The present invention relates to biological field, be specifically related to a kind of detection method protein-interacting.
Background technology:
Immunoprecipitation (immunoprecipitation, the method that IP) to be the specificity of utilizing antigen and antibody develop in conjunction with (for example bacterioprotein " albumin A " and immunoglobulin FC fragment specifically in conjunction with).For example, the antibody A immunoprecipitation X with albumin X may also precipitatedly get off with the protein Y that X combines in vivo so.Based on interacting with the physiological of albumin X, the immunoprecipitation of protein Y just cry co-immunoprecipitation (co-immunoprecipitation, Co-IP).
Co-immunoprecipitation is to find or verify physiological effective, the most the most frequently used interactional method between two kinds of protein.Its concrete operations are to keep albumen-protein-interacting (protein-proteininteraction, PPI) results and cell lysis under the condition, immunoprecipitation destination protein from cell extract is then by polyacrylamide gel electrophoresis (SDS-PAGE) separating immune sediment.Many at present albumin A combinations in advance with purifying are fixed on agarose (argarose) magnetic bead (beads), make it with antigen, antibody response after the cell pyrolysis liquid effect, the antigen of albumen (prorein) A on the magnetic bead (beads) in just can the adherent cell lysate, thus reach the purpose of precipitation target proteins (antigen) and albumen interactional with it.Further detect interaction protein again by SDS-PAGE separation, Western blotting (Western blot).
Traditional co-IP is as detecting and identify the most classical and effective method of PPI, in functional genome research, brought into play enormous function, yet this method itself also exists some significantly deficiencies: loaded down with trivial details as complicated operating process, waste time and energy, and often need to cultivate a large amount of cells.And, detect interaction by SDS-PAGE separating immune sediment, Western blot and further prolonged experimentation, limit the efficient that detects largely, thereby do not met the requirement of high throughput testing.
On the other hand, in the current protein group epoch, correlative study progressively deeply, need mass data information as analysis and research protein the basis.Traditional co-IP detects authentication method can not satisfy its needs.Protein-chip (protein array) is a kind of effective high flux research method of rising in recent years.It is similar to genetic chip, is that protein is fixing to solid phase carrier, and tissue that detects with need or cell etc. are discerned specifically mutually then, obtain a result by the self-reacting device analysis again.Compare with traditional research method, the advantage of protein-chip is that miniaturization and high pass quantize, can the thousands of protein sample of parallel analysis, have very high sensitivity and accuracy.Yet, existing protein chip, antibody chip as the release of U.S. BD Clontech company, be monoclonal antibody (the Ab Microarray380 that on chip, has arranged 378 kinds of known proteins, catalog number (Cat.No.) K1847-1), by immune response, in experiment, filter out the albumen that is associated with one or more monoclonal antibodies wherein.This chip only by the reaction between immune response detection monoclonal antibody and the albumen, thereby need be clicked and entered multiple monoclonal antibody.Its major defect is the antibody that must obtain various albumen, and present obtainable antibody is very limited, is difficult to satisfy the requirement of current proteomics research.And the present invention utilizes the epi-position label of fusion, uses universal antibody to prepare antibody chip, carries out the detection of any two kinds of protein-interactings on chip, has overcome the unobtainable bottleneck problem of antibody in the antibody chip preparation.
Summary of the invention:
The objective of the invention is to the co-immunoprecipitation reaction is combined application with protein chip, utilize chip technology to set up the method for the high throughput analysis albumen-protein-interacting (PPI) of a kind of simple and direct practicality, rapid sensitive.
The invention provides a kind of method that detects protein-interacting based on the co-immunoprecipitation of protein chip, wherein, prepare a Flag antibody chip, the cell pyrolysis liquid that will contain Flag-bait fusion protein and Myc-prey fusion then joins chip surface and carries out the co-immunoprecipitation reaction, whether has interaction through fluorescence labeling c-Myc antibody test bait protein and prey albumen.
Co-immunoprecipitation based on protein chip provided by the invention detects the method for protein-interacting, adopts following steps:
Step 1: preparation Flag antibody chip, aldehyde slide is divided into a plurality of chip frames, with anti-flag M2 monoclonal antibody and as the mouse IgG specking of antibody control in each chip frame, preserve standby;
Step 2: make up bait and prey expression vector, the encoding gene of bait is building up on the flag label carrier, the encoding gene of prey is building up on the Myc label carrier, form bait fusion protein and prey fusion respectively;
Step 3: cell transfecting and preparation lysate, in cell, collecting cell and fully cracking are collected experiment product cell pyrolysis liquid supernatant stand-by with bait and prey carrier for expression of eukaryon cotransfection; Same preparation negative control product cell pyrolysis liquid is collected stand-by;
Step 4: detect protein-interacting, in different chip frames in experiment product cell pyrolysis liquid and the chip after the reference substance cell pyrolysis liquid adds processing respectively, behind the co-immunoprecipitation reaction certain hour, washing; In each chip frame, add anti-c-Myc-Cy3 again, wash after reaction a period of time, dry back fluorescence chip scanner scanning;
Step 5: with the signal and the data output of obtaining in the fluorescence chip scanner, the fluorescence signal of comparative experiments product and reference substance, the experiment product signal intensity is better than reference substance, illustrate between bait and the prey albumen interaction is arranged, otherwise, do not have not interact between the explanation bait of signal or weak output signal and the prey albumen.
The method that detects protein-interacting based on the co-immunoprecipitation of protein chip provided by the invention, the Flag antibody chip is divided into 3 * 6 chip frames with pad pasting in the step 1, parallelly in each chip frame click and enter the anti-flagM2 monoclonal antibody and as the mouse IgG of antibody control, anti-flag M2 monoclonal antibody and 3 of mouse IgG each points repeat a little.
Co-immunoprecipitation based on protein chip provided by the invention detects the method for protein-interacting, in the step 4:
The chip processing is meant with confining liquid seals chip, and incubated at room is used the TBST repeated washing then;
With the amount in experiment product cell pyrolysis liquid and the reference substance cell pyrolysis liquid adding chip frame is 20 μ l/ frames;
Described co-immunoprecipitation reaction refers to react 2 hours under room temperature is preserved moisture condition;
Described washing refers to repeat 3 times with TBST washing 15 minutes;
The concentration of described adding anti-Myc-Cy3 monoclonal antibody is 1:200, and addition is 20 μ l/ chip frames, is reflected at the reaction 1 hour of preserving moisture under the condition of room temperature lucifuge.
The present invention also provides a kind of reagent kit for detecting protein interaction, comprises that one has the aldehyde slide of a plurality of subregions, FLAG in the said method and c-Myc label carrier and its corresponding monoclonal antibody, and based on the operation instructions of said method.
Aldehyde slide is divided into 3 * 6 subregions in the kit provided by the invention.
In the kit provided by the invention, the aldehyde slide model is CSS-100, and the FLAG label carrier is the pFLAG-CMV-2 of SIGMA company, and the c-Myc label carrier is that (pCMV-Myc of CLONTECH company, FLAG monoclonal antibody are SIGMA company
M2 monoclonal antibody, c-Myc monoclonal antibody are the monoclonal ANTI-c-MYC Cy3CONJUGATE CLONE 9E10 of SIGMA company.
Adopt above technical scheme, the present invention utilizes the epi-position label of recombinant protein, the universal antibody of anti-label is fixed on the aldehyde slide, make it catch target proteins in the cell pyrolysis liquid, under the condition that keeps albumen-protein-interacting, by the albumen of fluorescently-labeled antibody test co-immunoprecipitation, obtain the signal of albumen-protein-interacting at last by the fluorescence chip scanner.Its maximum characteristics are that miniaturization and high pass quantize, can a large amount of samples of parallel analysis, have very high susceptibility and accuracy, and a simplified control, but specific efficient detects the interaction between the albumen and albumen in the cell pyrolysis liquid.Compare with existing antibody chip, it does not need to prepare or buy the specific antibody of various albumen, and only need two kinds of general anti-tag antibodies just can carry out the detection of any two kinds of albumen interphase interactions, overcome the unobtainable bottleneck problem of antibody in the antibody chip preparation.On the other hand, compare with other protein chips that are used for the protein-interacting detection, it does not need to carry out the expression and purification of various albumen, the detection and the cell pyrolysis liquid that directly utilizes bait and prey cotransfection interacts, overcome the bottleneck problem of dietary protein origin difficulty in the protein chip preparation, both time saving and energy saving, save research cost again greatly.
Description of drawings:
Fig. 1 is the inventive method experiment flow synoptic diagram.
Fig. 2 is the chip co-immunoprecipitation of the present invention and the detection synoptic diagram that interacts.
Fig. 3 identifies figure for the double digestion electrophoresis of pCMV-myc-p65 among the present invention and pFlag-p50 recon, and wherein, each swimming lane is represented: 1.pCMV-myc-p65 (enzyme is not cut); (2.pCMV-myc-p65 xhoI and NotI enzyme are cut); (3.pFlag-p50 enzyme is not cut); (4.pFlag-p50 EcoRI and SalI enzyme are cut)
Fig. 4 is a co-immunoprecipitation chip detection p65-p50 interaction fluorescence signal picture of the present invention.
Fig. 5 is the potential interactional fluorescence signal picture of 6 pairs of albumen of co-immunoprecipitation chip checking of the present invention.
Fig. 6-1 and Fig. 6-2 is potential interactional fluorescence intensity of 6 pairs of albumen of co-immunoprecipitation chip checking of the present invention and ratio figure thereof.Fig. 6-1 is the interactional fluorescence intensity of sample: 1, and 2:FN1 and negative control thereof; 3,4:Myo18A and negative control thereof; 5,6:ATF5 and negative control thereof; 7,8:HLA-B and negative control thereof; 9,10:ATF4 and negative control thereof; 11,12:MCM3AP and negative control thereof.Fig. 6-2 is the interaction fluorescence intensity ratio of sample: the 1:FN1/ negative control; The 2:Myo18A/ negative control; The 3:ATF5/ negative control; The 4:HLA-B/ negative control; The 5:ATF4/ negative control; The 6:MCM3AP/ negative control.
Fig. 7 is 5 pairs of potential interactional western blot figures of albumen of traditional co-immunoprecipitation checking.
Embodiment:
Below describe the present invention in detail with specific embodiment.
The used reagent of the present invention comprises:
Antibody: monoclonal anti FLAG antibody (Monoclonal anti-
M2antibody, Sigma); Cy3 mark monoclonal anti c-Myc antibody (Monoclonal anti-c-Myc-Cy3antibody, Sigma); Mouse IgG (middle mountain gold bridge biotech firm).
Liposome: liposome 2000 (Lipofectamine
TM2000reagent, Invitrogen).
Aldehyde slide: the CSS-100 aldehyde slide (Aldehyde CSS-100Silylated Slides, CELAssociates, Inc.)
Protease inhibitors: no EDTA type adequate proteins enzyme inhibitor tablet (complete mini, EDTA free, protease inhibitor cocktail tablets, Roche)
Other various molecular biology common agents are commercially available import reagent.
The reagent preparation:
EBC lysis buffer: Tris-Cl 50mmol/L, pH8.0
NaCl 120mmol/L
NP-40 0.5％(V/V)
EDTA 1mmol/L
With preceding adding 50 μ g/ml PMSF, protease inhibitors
TBS： Tris-Cl 20mmol/L，pH8.0
NaCl 150mmol/L
TBST: the TBS that contains 0.05% (V/V) polysorbas20
Confining liquid: 10mg/ml BSA (bovine serum albumin(BSA)) is dissolved among the TBST.
The present invention detects the interaction between bait and the prey albumen, can adopt following operation, referring to flow process shown in Figure 1 and principle shown in Figure 2:
The preparation of Flag antibody chip: on aldehyde slide, stick pad pasting, make its subregion form 3 * 6 chip frames that are used to distinguish sample.In each subregion frame of aldehyde radical glass sheet, 3 of parallel points contain the point of anti-flag M2 monoclonal antibody and the point that contains mouse IgG of respective amount in each reaction frame with anti-flag M2 monoclonal antibody and mouse IgG (antibody in contrast) specking.4 ℃ of preservations are standby.
The embodiment of the invention adopts the M2 monoclonal antibody, because the M2 monoclonal antibody is a mouse IgG, therefore adopts mouse IgG as antibody control.The invention is not restricted to adopt the M2 monoclonal antibody, also can select the monoclonal antibody of other models for use.
The structure of bait and prey expression vector: the encoding gene of bait is building up on the flag label carrier, the encoding gene of prey is building up on the Myc label carrier, form flag-bait fusion protein and Myc-prey fusion respectively.
The preparation of cell transfecting and lysate: select cultured cell, when cellular incubation during to 80% density, with bait and prey carrier for expression of eukaryon cotransfection, collecting cell and fully cracking are collected experiment product cell pyrolysis liquid supernatant stand-by with liposome.With prepare the reference substance cell pyrolysis liquid with quadrat method.
Chip CoIP and interactional detection: confining liquid sealing chip, one of therein add the experiment product cell pyrolysis liquid in the chip frame after the washing, in another chip frame, add the reference substance cell pyrolysis liquid simultaneously as negative control, behind the reaction certain hour, washing; In each chip frame, add fluorescently-labeled another monoclonal antibody (anti-c-Myc-Cy3) again, wash after reaction a period of time, pad pasting on the chip is taken off, treat to put into fluorescence chip scanner picked up signal and reading of data after the slide drying, fluorescence signal is arranged, illustrating between two albumen has interaction, does not have the explanation of signal or weak output signal not interact.
Embodiment 1: the p65 of nuclear transcription factor-kappa B (NF-κ B) and the interactional co-immunoprecipitation chip analysis of p50 subunit
The p65 of NF-κ B and p50 subunit exist with the form of heterodimer under physiological condition, therefore, and with the positive design present embodiment of the interaction of p65 and p50.
One, the preparation of Flag antibody chip: on aldehyde slide, stick pad pasting, make its subregion form 3 * 6 chip frames.With microarray sample applicator (microarrayer, CAPITALBIO is rich difficult to understand, brilliant core SmartArrayer-48) with anti-flag M2 monoclonal antibody and mouse IgG (antibody in contrast) specking to each subregion frame of aldehyde radical glass sheet in, put 3 point and 3 points that contain mouse IgG that contain anti-flag M2 monoclonal antibody in each reaction frame.4 ℃ of preservations are standby.
Two, the structure and the evaluation of bait and prey expression vector: p65 (NM_021975) and p50 (NM_003998) are building up to respectively on pCMV-Myc and the pCMV-Flag-2 carrier, obtain pCMV-myc-p65 and pFLAG-p50 expression vector.
The building process concrete operations:
A) design p65 and p50 Auele Specific Primer, company is synthetic by the match Parkson:
Primer title primer sequence restriction enzyme
P50-up 5 '-CCGC
GAATTCAATGGCAGAAGATGATCC-3 ' (sequence 1) EcoRI
P50-down 5 '-CAGA
GTCGACCTAAGTGTCCATGGTTCC-3 ' (sequence 2) SalI
P65-uD 5 '-ATCT
CTCGAGGTATGGACGAACTGTTCCCC-3 ' (sequence 3) XhoI
P65-down 5 '-CAAT
GCGGCCGCTTAGGAGCTGATCTGACTC-3 ' (sequence 4) NotI
B) be template with liver cDNA library (Invitrogen), pcr amplification p65 and p50 gene: the PCR system is: 10 * Buffer5 μ l, dNTP Mixture (2.5mM) 4 μ l, each 1 μ l of synthetic primer (10 μ M) p50-up, p50-down, Pyrobest Taq (5U/ μ l) 0.5 μ l, cDNA library 1 μ l adds deionized water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ of extension 2min, 35 circulations of increasing; 72 ℃ are continued to extend 7min.
C) the PCR product carries out 1% agarose gel electrophoresis, and uviol lamp downcuts purpose band (the p65 size is 1656bp, and the p50 size is 1314bp) down, uses gel recovery kit (Promega) and reclaims genes of interest according to the described method of its instructions.
D) genes of interest and respective carrier are carried out restriction enzyme digestion: p50 and pFLAG-CMV-2 EcoRI﹠amp; SalI carries out double digestion, p65 and pCMV-Myc XhoI﹠amp; NotI carries out double digestion, and reaction system is as follows:
10×H?buffer 5μl
PCR purified product P50 (or P65) 20 μ l
EcoRI (or XhoI) 2.5 μ l
SalI (or NotI) 2.5 μ l
Add sterilized water to 50 μ l, 37 ℃ of water-bath enzymes are cut 4h.
E) enzyme is cut product and is carried out 1% agarose gel electrophoresis, use gel reclaim kit (
SV Geland PCR Clean-Up System Promega) and according to the described method of its instructions reclaims endonuclease bamhi, downcuts genes of interest (P50 or P65) and carrier endonuclease bamhi (pFLAG-CMV-2 or pCMV-Myc).
F) connect with the T4 ligase, linked system is as follows:
10×Buffer 2μl
Genes of interest 10 μ l
T4DNA?Ligase 1μl
Add deionized water to 20 μ l, room temperature connects 2 hours or 4 ℃ of connections are spent the night.
G) connect product transformed into escherichia coli JM109 competent cell: 20 μ l are connected product add in the 100 μ l escherichia coli jm109 competent cells, rotate mixing gently; Ice-water bath 30 minutes; 42 ℃ of heat shocks 90 seconds; Placed on ice 2 minutes; Add 500 μ l LB nutrient solutions, 37 ℃ of shaking tables (150rpm) were cultivated 45 minutes; Nutrient solution is applied on the LB solid medium flat board that is added with the ammonia benzyl; After drying up in the superclean bench, 37 ℃ of incubators are inverted overnight incubation.
Adopt following steps that pCMV-Myc-p65 and pFLAG-p50 carrier are identified:
H) picking monoclonal bacterium colony to is added with 3～5ml and contains the aseptic glass test tube of LB fluid nutrient medium of ammonia benzyl from flat board, and 37 ℃ of shaking tables (200rpm) spend the night.
I) use in a small amount plasmid DNA extract kit (
Plus SV Minipreps is Promega) and according to the described method upgrading of its instructions grain.
J) plasmid carries out double digestion evaluation recon: pFLAG-p50 EcoRI﹠amp; SalI carries out double digestion, pCMV-Myc-p65 XhoI﹠amp; NotI carries out double digestion, and reaction system is as follows:
10×H?buffer 2μl
The plasmid DN 5 μ l that contain recon pFLAG-p50 (or pCMV-Myc-p65)
EcoRI (or XhoI) 1 μ l
SalI (or NotI) 1 μ l
Add sterilized water to 20 μ l, 37 ℃ of water-bath enzymes are cut 4h.
K) enzyme is cut product and is carried out 1% agarose gel electrophoresis, if electrophoresis result shows size and corresponding to two bands of expection, shows that then this plasmid is a recon.The result is as shown in Figure 3: 2,4 recons that are respectively pCMV-myc-p65 and pFlag-p50 wherein.
1) selects the recon affirmation of checking order.
Three, the preparation of cell transfecting and lysate:
1, the preparation of pCMV-myc-p65 and pFLAG-p50 cotransfection cell pyrolysis liquid (experiment product)
With the high sugared nutrient solution of the DMEM that contains 10%FBS (hyclone) (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.) (the kind Bioisystech Co., Ltd that reaches of Beijing Tian Run), containing 5%C0
237 ℃ of incubators in cultivate the HEK-293 cell (HEKC be inoculated on the 24 porocyte culture plates, China Concord Medical Science University's Institute of Basic Medical Sciences's cell centre), the microscopically observation of cell, when cell long during to about 80% density, with liposome 2000 with pCMV-myc-p65 and pFLAG-p50 cotransfection cell: in an aseptic EP pipe of 0.5ml, add the DMEM nutrient culture media of 300ng pCMV-myc-p65,300ng pFLAG-p50 and 50 μ l serum-frees simultaneously, mixing; The DMEM nutrient culture media that in the aseptic EP pipe of another 0.5ml, adds 2 μ l liposomes 2000 and 50 μ l serum-frees simultaneously, mixing; After the incubated at room 5 minutes, both are admixed together, and incubated at room is 20 minutes behind the mixing; Said mixture is added in each porocyte of 24 porocyte culture plates; Cell put back to contain 5%CO
237 ℃ of incubators in cultivate.
Collecting cell after transfection 24-36 hour: cell culture fluid is siphoned away, then with the PBS (phosphate buffer, 137mmol/L NaCl, 2.7mmol/L KCl, the 10mmol/L Na that are pre-chilled to 4 ℃
2HPO
4, 2mmol/LKH
2PO
4, pH7.4) clean cell, in each hole of 24 porocyte culture plates, add EBC lysis buffer (50 μ l/ hole) afterwards, room temperature is placed and was carried out abundant cracking in 15 minutes.Cell pyrolysis liquid in 24 each hole of porocyte culture plate is recovered in the EP pipe, and 4 ℃, centrifugal 10 minutes of 12000rpm, collecting cell lysate supernatant put into another clean EP pipe, and be standby as experiment product.
2, the preparation of pCMV-myc-p65 and pFLAG-CMV-2 cotransfection cell pyrolysis liquid (reference substance one)
With preparing reference substance one with 1 identical operations, wherein with liposome 2000 with pCMV-myc-p65 and pFLAG-CMV-2 cotransfection, as the checking the specific negative control of immunoprecipitation (flag-p50 disappearance).
3, the preparation of pCMV-Myc and pFLAG-p50 cotransfection lysate (reference substance two)
With preparing reference substance two with 1 identical operations, wherein with liposome 2000 with pCMV-Myc and pFLAG-p50 cotransfection, detect antibody (being the c-myc antibody of Cy3 mark) specific negative control (myc-p65 disappearance) as checking.
Four, chip CoIP and interactional detection:
With confining liquid (20 μ l/ frame) sealing chip, incubated at room 45 minutes; TBST washing 5 minutes repeats 3 times; In the first chip frame, add experiment product cell pyrolysis liquid (20 μ l/ frame), in the second chip frame, add reference substance one cell pyrolysis liquid (20 μ l/ frame), in the 3rd chip frame, add reference substance two cell pyrolysis liquids (20 μ l/ frame); Room temperature is preserved moisture and was reacted 2 hours; TBST washing 15 minutes repeats 3 times; Adding anti-Myc-Cy3 monoclonal antibody (1:200＜antibody: confining liquid 〉, 20 μ l/ chip frames), the room temperature lucifuge is preserved moisture and was reacted 1 hour; TBST washing 15 minutes repeats 3 times; Pad pasting on the chip is taken off, treat to put into fluorescence chip scanner (LuxScan-10K/A, CapitalBio) middle picked up signal and reading of data after the slide drying.
Five, testing result:
Fig. 4 has shown the interactional fluorescence signal of detection.As can be seen from the results: be fixed on flag-p50 under the anti-flag monoclonal antibody energy immunoprecipitation on the chip, and myc-p65 is because of being got off by co-immunoprecipitation with the interaction of p50, so can detect strong signal (referring to the capable A row of flag among Fig. 4).Mouse IgG as antibody control does not almost have signal (capable referring to IgG among Fig. 4), and simultaneously ((referring to the capable B row of flag among Fig. 4) and pCMV-Myc and pFLAG-p50 (referring to the capable C row of flag among Fig. 4) cotransfection cell pyrolysis liquid signal is also very weak as the pCMV-myc-p65 of sample contrast and pFLAG-CMV-2.Illustrate that co-immunoprecipitation chip of the present invention can effectively detect p65 and p50 interaction between protein, adopt mouse IgG as antibody control, illustrate that the compound under the flag antibody mediated immunity precipitation is the specificity product on the chip, therefore detected signal is a specificity target interaction between protein, gets rid of the possibility of non-specific adsorption.
Embodiment 2: the co-immunoprecipitation chip carries out interactional checking to six pairs of potential interactional albumen of tool:
Present embodiment utilizes the co-immunoprecipitation chip that the albumen in six pairs of yeast two-hybrid sources has been carried out the checking that interacts.
As bait, through yeast two-hybrid screening people liver cDNA library, obtain 6 prey albumen: FN1, Myo18A, ATF5, ATF4, MCM3AP and HLA-B with TRB3 albumen (triblles3) early stage.
Bait is building up on the pFLAG-CMV-2 carrier for expression of eukaryon (please refer to the method for embodiment 1), six kinds of preys are building up to respectively on the pCMV-Myc carrier for expression of eukaryon (please refer to the method for embodiment 1).
With reference to the method among the embodiment 1; With six kinds of pCMV-Myc-prey albumen respectively with pFLAG-TRB3 cotransfection HEK-293 cell, simultaneously with six kinds of pCMV-Myc-preys respectively with the negative control of pFlag-CMV-2 cotransfection as bait disappearance protein-interacting.After obtaining 12 cell pyrolysis liquids, carry out the co-immunoprecipitation reaction respectively in the different chip frames on chip, after fluorescent-labeled antibody anti-Myc-Cy3 monoclonal antibody detected, signal was accordingly interacted.
Testing result is referring to Fig. 5 and Fig. 6.Among Fig. 5, the prey title of transfection in each row of line display, the bait title of each row transfection is shown in tabulation, as first row: go up hole represented cotransfection myc-ATF5 and Flag-TRB3, cotransfection has been represented in following hole myc-ATF5 and pFlag-CMV-2); Show among the figure: prey ATF4, FN1, ATF5, MCM3AP and bait TRB3 have interaction, its fluorescence intensity is respectively 68.7,6.6,2.5,5.4 times of corresponding negative control, and prey Myo18A, HLA-B and bait TRB3 do not have tangible interaction signal, its fluorescence intensity is suitable with corresponding negative control, only 1.5 times.
The testing result of present embodiment is consistent (wherein based on the co-immunoprecipitation checking result (referring to Fig. 7) of resin with tradition, because mcy-HLA-B does not detect expression, therefore do not do itself and the co-immunoprecipitation analysis of TRB3), show that further the co-immunoprecipitation chip method that the present invention sets up is effective in analyzing proteins interacts.
By above embodiment explanation, the present invention combines protein-chip with CoIP, utilize FLAG and c-Myc epi-position label and its corresponding monoclonal antibody, has set up a kind of method that detects protein-interacting based on the co-immunoprecipitation of chip.This method makes cell pyrolysis liquid directly carry out the CoIP reaction at chip surface, behind the albumen of the non-specific bond of flush away, adopt fluorescently-labeled antibody directly to carry out interactional detection, save many time-consuming, trivial step such as precipitation, SDS-PAGE separation, western blot detection of the agarose magnetic bead of traditional C oIP, simplified experimentation greatly.In addition, use this method, sample, reagent demand significantly reduce, and same sample can carry out the parallel laboratory test of control antibodies simultaneously, reduce experimental cost greatly.And this method has made full use of that the chip high pass quantizes, characteristics such as flow process is simple, weak point consuming time, thereby can realize the flux of sample is detected.Carrying out protein-interacting when detecting, good reproducibility, background noise is little, and is highly sensitive and detection efficiency is suitable with traditional co-IP method.The inventive method can replace the classic method that takes time and effort in the past, becomes efficient strong instrument in the proteomics research.
The present invention is in concrete enforcement, can also form a kind of reagent kit for detecting protein interaction, this kit can comprise: the aldehyde slide (each aldehyde radical sheet can carry out the detection of 18 samples, and the interaction quantity that can detect is as required selected the quantity of aldehyde radical sheet) with a plurality of subregions; FLAG label carrier pFLAG-CMV-2; C-Myc label carrier pCMV-Myc; Monoclonal anti FLAG antibody; Cy3 mark monoclonal anti c-Myc antibody; Mouse IgG and operation instructions.Concrete a kind of kit, wherein the aldehyde slide model is CSS-100, and the FLAG label carrier is the pFLAG-CMV-2 of SIGMA company, and the c-Myc label carrier is that (pCMV-Myc of CLONTECH company, FLAG monoclonal antibody are SIGMA company
M2 monoclonal antibody, c-Myc monoclonal antibody are the monoclonal ANTI-c-MYC Cy3 CONJUGATE CLONE 9E10 of SIGMA company.
Claims (6)
1. method that detects protein-interacting based on the co-immunoprecipitation of protein chip, it is characterized in that, prepare a Flag antibody chip, the cell pyrolysis liquid that will contain Flag-bait fusion protein and Myc-prey fusion then joins chip surface and carries out the co-immunoprecipitation reaction, whether has interaction through fluorescence labeling c-Myc antibody test bait protein and prey albumen; The concrete following steps that adopt:
Step 1: preparation Flag antibody chip, aldehyde slide is divided into a plurality of chip frames, with anti-flag M2 monoclonal antibody and as the mouse IgG specking of antibody control in each chip frame, preserve standby;
Step 2: make up bait and prey expression vector, the encoding gene of bait is building up on the flag label carrier, the encoding gene of prey is building up on the c-Myc label carrier, form bait fusion protein and prey fusion respectively;
Step 3: cell transfecting and preparation lysate, in cell, collecting cell and fully cracking are collected experiment product cell pyrolysis liquid supernatant stand-by with bait and prey carrier for expression of eukaryon cotransfection; Same preparation negative control product cell pyrolysis liquid is collected stand-by;
Step 4: detect protein-interacting, in different chip frames in experiment product cell pyrolysis liquid and the chip after the reference substance cell pyrolysis liquid adds processing respectively, behind the co-immunoprecipitation reaction certain hour, washing; In each chip frame, add anti-c-Myc-Cy3 again, wash after reaction a period of time, dry back fluorescence chip scanner scanning;
Step 5: with the signal and the data output of obtaining in the fluorescence chip scanner, the fluorescence signal of comparative experiments product and reference substance, the experiment product signal intensity is better than reference substance, illustrate between bait and the prey albumen interaction is arranged, otherwise, do not have not interact between the explanation bait of signal or weak output signal and the prey albumen.
2. according to the described method of claim 1, it is characterized in that, the Flag antibody chip is divided into 3 * 6 chip frames with pad pasting in the described step 1, parallelly in each chip frame click and enter anti-flag M2 monoclonal antibody and as the mouse IgG of antibody control, anti-flag M2 monoclonal antibody and 3 of mouse IgG each points repeat a little.
3. according to claim 1 or 2 described methods, it is characterized in that, in the described step 4,
The chip processing is meant with confining liquid seals chip, and incubated at room is used the TBST repeated washing then;
With the amount in experiment product cell pyrolysis liquid and the reference substance cell pyrolysis liquid adding chip frame is 20 μ l/ frames;
Described co-immunoprecipitation reaction refers to react 2 hours under room temperature is preserved moisture condition;
Described washing refers to repeat 3 times with TBST washing 15 minutes;
The concentration of described adding anti-c-Myc-Cy3 monoclonal antibody is 1: 200, and addition is 20 μ l/ chip frames, is reflected at the reaction 1 hour of preserving moisture under the condition of room temperature lucifuge.
4. reagent kit for detecting protein interaction, it is characterized in that, comprise that one has the aldehyde slide of a plurality of subregions, flag described in the arbitrary described method of claim 1 to 3 and c-Myc label carrier and its corresponding monoclonal antibody, and based on the operation instructions of the arbitrary described method of claim 1 to 3.
5. kit according to claim 4 is characterized in that, described aldehyde slide is divided into 3 * 6 subregions.
6. according to claim 4 or 5 described kits, it is characterized in that described aldehyde slide model is CSS-100, described flag label carrier is the pFLAG-CMV-2 of SIGMA company, described c-Myc label carrier is the pCMV-Myc of CLONTECH company, and described flag monoclonal antibody is a SIGMA company
Monoclonal antibody, the monoclonal ANTI-c-MYC Cy3 CONJUGATE CLONE 9E10 that described c-Myc monoclonal antibody is a SIGMA company.
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| CN107051397A (en) * | 2017-02-23 | 2017-08-18 | 南昌大学 | The affine in immunity sorbing material of specific recognition c Myc label nano antibodies |
| CN107042092A (en) * | 2017-02-23 | 2017-08-15 | 南昌大学 | Affine sorbing material based on specific recognition c Myc label single domain heavy chain antibodies |
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