CN109470756A - Measure the electrochemical method of 14-3-3 albumen and plasmalemma H+-ATPase interactions between protein - Google Patents
Measure the electrochemical method of 14-3-3 albumen and plasmalemma H+-ATPase interactions between protein Download PDFInfo
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- 102000015176 Proton-Translocating ATPases Human genes 0.000 title 1
- 108010039518 Proton-Translocating ATPases Proteins 0.000 title 1
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- 108091006112 ATPases Proteins 0.000 claims abstract description 39
- 102000057290 Adenosine Triphosphatases Human genes 0.000 claims abstract description 39
- 239000000523 sample Substances 0.000 claims abstract description 26
- 238000007650 screen-printing Methods 0.000 claims abstract description 25
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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Abstract
The invention discloses a kind of measurement 14-3-3 albumen and plasma membrane H+The electrochemical method of-ATPase interactions between protein selects screen printing electrode with good conductivity, by capture probe plasma membrane H+- ATPase antibody is fixed on the working electrode of screen printing electrode by physical absorption, after 14-3-3 albumen and signal probe association reaction in albumen composition to be detected, horseradish peroxidase-labeled Streptavidin and signal probe are connected to surface of printing electrode, and realize detection using the redox current of electrochemical analyser detection horseradish peroxidase enzyme catalytic substrate；14-3-3 albumen and plasma membrane H can be fast implemented using this electrochemical method+The highly sensitive detection of-ATPase interactions between protein；The advantages that detection method has high sensitivity, cheap, and detection speed is fast, can be used for 14-3-3 albumen and plasma membrane H+The detection of-ATPase interactions between protein.
Description
Technical field
The invention belongs to electrochemical analysis techniques field, especially 14-3-3 albumen and plasma membrane H+- ATPase albumen
The electrochemical method of interaction.
Background technique
Plant 14-3-3 albumen can be by adjusting in plant with the interaction of the protein of phosphorylation or non-phosphorylating
A variety of bioprocess, such as synthesis/folding, primary metabolite, signal transduction and the hormone metabolism of protein etc..There is result of study
Show plasma membrane H+The activity of-ATPase is mainly by the adjusting in its area CDuan Ziyi, 14-3-3 albumen and plasma membrane H+- ATPase phosphoric acid
The C-terminal of change is combined from inhibition zone, activates proton pump；And the plasma membrane H on plasmalemma of plant+- ATPase is 14-3-3 albumen
Main function target spot, 14-3-3 albumen is in regulation plasmalemma of plant H+It is played an important role in the activity of-ATPase.
Currently, co-immunoprecipitation be it is a kind of study protein-interacting classical way, if albumin A in conjunction with protein B,
So when we remove binding protein A with antibody, protein B, which can be also pulled down into, to be come；Vice versa.But this method is deposited
More complex in operating procedure, detection limit is higher, the problems such as poor selectivity.
Currently, the detection of interactions between protein mainly passes through Immunoprecipitation, pull-down technology, yeast two-hybrid
System, display technique of bacteriophage etc., but these technologies the disadvantages of there is cumbersome, sensitivity is low, expensive.
Summary of the invention
It is an object of that present invention to provide a kind of measurement 14-3-3 albumen and plasma membrane H+The electrochemistry side of-ATPase interactions between protein
Method, the method for the present invention are prepared on the basis of electrochemical analysis using the antibody of one of two kinds of interaction albumen as the electricity of capture probe
Chemical sensor establishes high sensitivity, the cheap and fireballing interactions between protein detection method of detection.
The method of the present invention the following steps are included:
(1) working electrode on screen printing electrode is activated, working electrode surface is made to generate carboxyl；
(2) 14-3-3 protein antibodies liquid is added dropwise on the working electrode of the screen printing electrode after step (1) activation, 4 DEG C combine 5
After~20h, with electrode washing lotion flushing work electrode 3~4 times, drying；Bovine serum albumin(BSA) fluid-tight is added dropwise again and closes active site, room
Temperature closing 0.5h~2h, electrode washing lotion flushing work electrode 3~4 times, is dried；
(3) 14-3- of the 5 μ L concentration within the scope of 0 μ of μ g/mL~12 g/mL is added dropwise on multiple working electrodes of step (2) drying
3 protein liquids, in conjunction with electrode washing lotion flushing work electrode after 1~2h, drying；
(4) the Biotin-14-3-3 antibody liquid of biotin labeling is added dropwise on the working electrode (s, is rushed in conjunction with electrode washing lotion after 1~2h
Working electrode is washed, is dried；
(5) horseradish peroxidase-labeled solution of streptavidin is added dropwise on the working electrode (s, reacts at room temperature 10~60min, uses
Electrode washing lotion is cleaned, and 3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions is added dropwise on screen printing electrode, then in electrification
Size of current is measured using cyclic voltammetry and current versus time curve method in credit analyzer, with 14-3-3 protein content and correspondence
Current value, make working curve, obtain equation of linear regression；
(6) plasma membrane H is added dropwise on the working electrode of the screen printing electrode after step (1) activation+- ATPase antibody liquid, as
Capture probe, after 4 DEG C combine 5~20h, the same step of subsequent operation (2), working electrode made from the step is electrochemical sensing
Device；The object to be detected extracted from plant root is added dropwise on electrochemical sensor, rinses electrochemistry in conjunction with electrode washing lotion after 1~2h
Sensor, to wash away the foreign protein of non-specific binding；The Biotin-14- of biotin labeling is added dropwise on electrochemical sensor
3-3 antibody rinses electrochemical sensor, drying in conjunction with electrode washing lotion after 1~2h as signal probe；It is surveyed by step (5) method
Constant current size brings the current value measured in equation of linear regression into, obtains and plasma membrane H+The 14-3-3 egg that-ATPase is combined
White amount.
The activation method of the working electrode is that 80 μ of μ L~200 L are added dropwise in the working electrode surface on screen printing electrode
Electrolyte solution, carry out cyclic voltammetry scan with electrochemical analyser, make working electrode surface generate carboxyl；Wherein electrolyte
Solution is the phosphate buffer of 8~15mmol/L of concentration, pH 7.4；The voltage of cyclic voltammetry scan is -0.3~0.4V, is swept
Speed is 500mV/s, scans 10 circles altogether.
The electrode washing lotion is the phosphate buffer of pH7.4, contains 0.05~0.2mmol/L in phosphate buffer
The Tween-20 that NaCl and mass concentration are 1%.
The 14-3-3 protein antibodies liquid is configured to concentration with the phosphate buffered solutions containing 1~2% bovine serum albumin(BSA)
The 18 μ g/mL of μ g/mL~30, dripping quantity are 3 μ of μ L -6 L.
The bovine serum albumin white liquor uses the phosphate buffered saline of pH 7.4, and mass concentration is 1~2%, dripping quantity
For 5~10 μ l.
The Biotin-14-3-3 antibody liquid of the biotin labeling is that Biotion-XX-NHS is dissolved in N, N- dimethyl
In formamide, then 14-3-3 antibody is dissolved in the phosphate buffer that 0.008~0.015mol/L pH is 7.4, it will be above-mentioned
After two solution mixing, 1~4h of reaction is stirred at room temperature, reaction solution crosses gel column, is with 0.008mol/L~0.015mol/LpH
7.4 phosphate buffer elution, collects eluent to get the Biotin-14-3-3 antibody of biotin labeling is arrived, wherein
The mass ratio of Biotion-XX-NHS and 14-3-3 albumen is 15-25:1.
The Biotin-14-3-3 antibody liquid concentration of the biotin labeling is the 25 μ g/mL of μ g/mL~200, and 3~6 μ are added dropwise
l。
The horseradish peroxidase-labeled solution of streptavidin is by horseradish peroxidase-labeled Streptavidin
It is dissolved in the phosphate buffered solutions containing 1~2% bovine serum albumin(BSA), concentration is the 0.5 μ g/mL of μ g/mL~3, and dripping quantity is 3~6
μL。
Plasma membrane H in the step (6)+- ATPase antibody liquid concentration is 18~30 μ g/mL, and dripping quantity is 3~6 μ L.
The 3,3', 5,5'- tetramethyl biphenyl amine aqueous solution in tmb substrate colour reagent box by illustrating to prepare.
14-3-3 albumen is that pGEX4T-14-3-3 prokaryotic expression carrier is transferred in e. coli bl21 in the present invention, induces table
It is obtained up to simultaneously affinity purification, 14-3-3 antibody is to be carried out three times by emulsification 14-3-3 albumen to adult male New Zealand rabbit
It is prepared after immune.
The plasma membrane H+- ATPase antibody is by pGEX4T-H+- ATPase prokaryotic expression carrier goes to e. coli bl21
In, inducing expression and affinity purification emulsify plasma membrane H+Adult male New Zealand rabbit is carried out after being immunized three times after-ATPase albumen
It obtains.
Screen printing electrode in the present invention is Conventional electrochemical electrode, and using carbon slurry as working electrode, silver chlorate is reference
Electrode, carbon slurry are to print electrode to pole.
The present invention selects screen printing electrode with good conductivity first, by capture probe -- plasma membrane H+- ATPase antibody
Be fixed on the working electrode of screen printing electrode by physical absorption, the 14-3-3 albumen in albumen composition to be detected and
After signal probe, that is, biotin labeling Biotin-14-3-3 antibody association reaction, horseradish peroxidase-labeled strepto- is affine
Element with biotin is affine is connected to surface of printing electrode, utilize electrochemical analyser detection horseradish peroxidase enzyme catalytic substrate
Redox current realizes detection, can fast implement 14-3-3 albumen and plasma membrane H using electrochemical method+- ATPase albumen is mutual
The highly sensitive detection of work；The advantages that detection method has high sensitivity, cheap, and detection speed is fast, can be used for 14-3-
3 albumen and plasma membrane H+The detection of-ATPase interactions between protein.
Compared with prior art, the present invention having the advantage that
(1) high specificity in reaction system of the invention, lacks any ingredient and all examines and do not measure curent change；
(2) high sensitivity, since 14-3-3 albumen and biotinylated 14-3-3 antibody specificity combination are very strong, horseradish peroxidating
Object enzyme can be with the redox of catalysis substrate, to make present invention sensitivity with higher；
(3) detection speed is fast, and the present invention is time-consuming short, and step is simple；
(4) selectivity is good, combines 1 hour every time, needs to be rinsed with electrode washing lotion, at this time capture probe and antigen, antigen and letter
Number probe is specifically bound, and the foreign protein and small-molecule substance of non-specific binding can be washed off, and the present invention is presented
Good selectivity；
(5) it quantitative determines, the present invention can be with quantitative detection plasma membrane H+The 14-3-3 protein content that-ATPase is combined.
Detailed description of the invention
Fig. 1 is that the linear relationship of current peak and different content 14-3-3 antigen and feasibility detect；A figure is current peak
With the linear relationship of different content 14-3-3 antigen, B figure is the I-T figure of different content 14-3-3 antigen；
Fig. 2 is the repeatability detection of current peak and different content 14-3-3 antigenic relationship；A figure is current peak and different content
The column diagram of 14-3-3 antigen repeatability detection, B figure are the I-T figure of three groups of different content 14-3-3 antigens；
Fig. 3 is plasma membrane H+- ATPase antibody makees capture probe, 14-3-3 albumen and plasma membrane H+The method of-ATPase interactions between protein
Verification result figure；
Fig. 4 is the plasma membrane H for the plasmalemma protein sample that aluminium handles Tobacco Root+- ATPase activity；A figure is different disposal time plasma membrane H+- ATPase combines 14-3-3 protein content, and B figure is the I-T figure of protein sample；
Fig. 5 is the plasma membrane H for the plasmalemma protein sample that aluminium handles Tobacco Root+- ATPase activity；
Fig. 6 is that the plasmalemma protein sample of aluminium processing Tobacco Root analyzes 14-3-3 albumen and plasma membrane H using co-immunoprecipitation+-
The interaction of ATPase albumen is horizontal, and wherein A is to analyze 14-3-3 albumen and plasma membrane H using co-immunoprecipitation+- ATPase albumen
Interaction is horizontal, and B is plasma membrane H+The opposite binding capacity of-ATPase, C are the opposite binding capacity of 14-3-3 albumen.
Specific embodiment
Invention is further described in detail in the following with reference to the drawings and specific embodiments, but protection scope of the present invention is simultaneously
It is not limited to the content.
Embodiment 1: the linear relationship and repeatability of current peak and recombinant 14-3-3 protein antigen detect
(1) 100 μ l 10mmol/L PBS(pH 7.4 are added dropwise in the working electrode surface on screen printing electrode), as electrolysis
Matter solution carries out cyclic voltammetry scan with CHI 660e electrochemical analyser to activate working electrode surface and generate carboxyl；Circulation
Voltammetric scan voltage range is -0.3~0.4V, and sweeping speed is 500mV/s, scans 10 circles altogether；
(2) the 14-3-3 protein antibodies that 5 μ l concentration are 25 μ g/mL are instilled on the working electrode of screen printing electrode (to use containing 1%
The phosphate buffered solutions of bovine serum albumin(BSA) are prepared), 4 DEG C combine 16h, with electrode washing lotion cleaning electrode three times and with red
Outer lamp drying, to wash away unadsorbed antibody；5 μ l mass concentration, 1% bovine serum albumin(BSA) room temperature closing 1h is instilled, is washed with electrode
Liquid cleaning electrode is dried three times and with infrared lamp；
(3) 14-3-3 albumen (antigen) is diluted with the PBS solution containing 1% bovine serum albumin(BSA), 5 μ L is added dropwise respectively to work electricity
On extremely, making the amount of antigen on working electrode is respectively 0ng, 1ng, 5ng, 10ng, 20ng, 25ng, 30ng, 40ng, 50ng,
60ng, wherein 0ng, 20ng, 35ng make three repetitions；Room temperature combination 1h, with electrode washing lotion cleaning electrode three times and with red
Outer lamp drying；
(4) after antigen-reactive, the Biotin- that 5 μ l concentration are 50 μ g/mL signal probe biotin labelings is added dropwise on the working electrode (s
14-3-3 antibody (is prepared) with the phosphate buffered solutions containing 1% bovine serum albumin(BSA), is reacted at room temperature 1h, is carried out with electrode washing lotion
It cleans and is dried with infrared lamp；
(5) it is that 1 μ g/mL horseradish peroxidase-labeled solution of streptavidin room temperature is anti-that 5 μ l concentration are added dropwise on the working electrode (s
It answers 30 minutes, is cleaned with electrode washing lotion；3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions are added dropwise on screen printing electrode,
Then size of current is measured using cyclic voltammetry and current versus time curve method on electrochemical analyser, with 14-3-3 albumen
Content and corresponding current value make working curve, obtain equation of linear regression y=109.32x+384.73；
As a result as shown in Figure 1, 2, Fig. 1 shows that 14-3-3 antibody is capture probe, and 14-3-3 albumen makees antigen, biotin labeling
When Biotin-14-3-3 antibody is signal probe, between antigenic content 0ng-60ng, current peak and antigenic content have well
Specificity and linear relationship；Fig. 2 shows that this method has good repeatability.
Embodiment 2: plasma membrane H+- ATPase antibody makees capture probe, the method validation of interactions between protein
(1) 100 μ l 10mmol/LPBS(pH7.4 are added dropwise in the working electrode surface on screen printing electrode), as electrolyte
Solution carries out cyclic voltammetry scan with CHI 660e electrochemical analyser to activate working electrode surface and generate carboxyl；Circulation volt
Peace scanning voltage is -0.3~0.4V, and sweeping speed is 500mV/s, scans 10 circles altogether；
(2) the plasma membrane H that 5 μ l concentration are 25 μ g/mL is instilled on the working electrode of screen printing electrode after activation+-ATPase
Antibody liquid, 4 DEG C combine 16h, are dried three times and with infrared lamp with electrode washing lotion cleaning electrode, to wash away unadsorbed resist
Body；Active site is closed in the bovine serum albumin(BSA) fluid-tight that 5 μ l mass concentrations 1% are added dropwise again, and room temperature is closed 1h, cleaned with electrode washing lotion
Working electrode is dried three times and with infrared lamp；
(3) by plasma membrane H+- ATPase phosphorylated polypeptide (antigen) is diluted with the phosphate buffered solutions containing 1% bovine serum albumin(BSA),
20ng plasma membrane H is added dropwise respectively on the working electrode of 3 screen printing electrodes+- ATPase phosphorylated polypeptide+20ng 14-3-3
Antigen, 20ng plasma membrane H+- ATPase phosphorylated polypeptide+0ng14-3-3 antigen；0ng plasma membrane H+- ATPase phosphorylated polypeptide+20ng
14-3-3 antigen, room temperature combination 1h are dried three times and with infrared lamp with electrode washing lotion cleaning electrode；
(4) after antigen-reactive, it is anti-that the Biotin-14-3-3 that 5 μ l concentration are 50 μ g/mL biotin labelings is added dropwise on the working electrode (s
Body fluid (is prepared) with the phosphate buffered solutions containing 1% bovine serum albumin(BSA), reacts at room temperature 1h, carries out work electricity with electrode washing lotion
Pole is cleaned and is dried with infrared lamp；
(5) it is that 1 μ g/mL horseradish peroxidase-labeled solution of streptavidin room temperature is anti-that 5 μ l concentration are added dropwise on the working electrode (s
It answers 30 minutes, is cleaned with electrode washing lotion；3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions are added dropwise on screen printing electrode,
Then size of current, each group of measurement 3 are measured using cyclic voltammetry and current versus time curve method on electrochemical analyser
It is secondary, it is averaged；The current value measured is brought into the equation of linear regression of embodiment 1, is obtained and plasma membrane H+- ATPase is combined
14-3-3 albumen amount；
As a result as shown in figure 3, being from left to right followed successively by 0ng plasma membrane H+- ATPase phosphorylated polypeptide+20ng14-3-3 antigen；
20ng plasma membrane H+- ATPase phosphorylated polypeptide+0ng14-3-3 antigen；For 20ng plasma membrane H+- ATPase phosphorylated polypeptide+20ng
14-3-3 antigen；Control is the result of 20ng antigen in embodiment 1；The result shows that 20ng plasma membrane H+- ATPase phosphorylation
The current peak of polypeptide+20ng 14-3-3 antigen and the current peak that 1 method of embodiment measures are almost consistent, illustrate plasma membrane H+-
ATPase antibody, which makees capture probe, can capture H+- ATPase-14-3-3 albumen composition, subsequent 14-3-3 proteantigen and letter
Number probe, that is, biotinylated 14-3-3 antibody association reaction, to realize current detecting.
Meanwhile by 20ng plasma membrane H+The current value of-ATPase phosphorylated polypeptide+20ng 14-3-3 antigen brings embodiment 1 into
Linear equation in, obtain and plasma membrane H+- ATPase combine 14-3-3 albumen amount be 19.957ng, about 20ng, more
Determine the feasibility of the method.
Embodiment 3: Zeeman GFAAS
The root of 0.5g wild-type tobacco is taken to be placed on containing 50 μm of ol/L AlCl first3、0.5mmol/L CaCl2, pH=4.3
0h, 2h, 8h and 12h are handled in solution, cleans and collect root system, and -80 DEG C of preservations (contain 0.25 M sorbierite, 1 with extract
Mmol/L EDTA, 10 mmol/L Tris-HCl(pH 7.4), 10 μm of ol/L NaF, 0.5 mmol/L PMSF, 0.015%(v/
V) Trix-100) extract plasmalemma protein；
(1) 150 μ l 10mmol/LPBS(pH7.4 are added dropwise in the working electrode surface on screen printing electrode), as electrolyte
Solution carries out cyclic voltammetry scan with CHI 660e electrochemical analyser to activate working electrode surface and generate carboxyl；Circulation volt
Peace scanning voltage is -0.3~0.4V, and sweeping speed is 500mV/s, scans 10 circles altogether；
(2) the plasma membrane H that 5 μ l concentration are 25 μ g/mL is instilled on the working electrode of screen printing electrode after activation+-ATPase
Antibody liquid, 4 DEG C combine 10h, are dried with electrode washing lotion cleaning electrode 4 times and with infrared lamp, to wash away unadsorbed antibody；
Active site is closed in the bovine serum albumin(BSA) fluid-tight that 5 μ l mass concentrations 1% are added dropwise again, and room temperature closes 1h, with electrode washing lotion cleaning
Electrode is dried three times and with infrared lamp, and working electrode is electrochemical sensor；
(3) the 5 μ l of plasmalemma protein extracted from Tobacco Root is added dropwise on the working electrode (s, rinses electrochemistry in conjunction with electrode washing lotion after 1h and passes
Sensor is dried with washing away the foreign protein of non-specific binding with infrared lamp；
(4) after antigen-reactive, the Biotin-14-3-3 that 5 μ l concentration are 50 μ g/mL biotin labelings is added dropwise on the working electrode (s
Antibody liquid (is prepared) with the phosphate buffered solutions containing 1% bovine serum albumin(BSA), is reacted at room temperature 1h, is worked with electrode washing lotion
Electrode clean is simultaneously dried with infrared lamp；
(5) it is that 1 μ g/mL horseradish peroxidase-labeled solution of streptavidin room temperature is anti-that 5 μ l concentration are added dropwise on the working electrode (s
It answers 30 minutes, is cleaned with electrode washing lotion；3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions are added dropwise on screen printing electrode,
Then size of current is measured using cyclic voltammetry and current versus time curve method on electrochemical analyser；The electric current that will be measured
Value is brought into the equation of linear regression of embodiment 1, is obtained and plasma membrane H+The amount for the 14-3-3 albumen that-ATPase is combined；
As a result as shown in figure 4, plasma membrane H when aluminium handles 0h+It is about 5.3ng/ μ g, aluminium processing that-ATPase, which combines 14-3-3 protein content,
The current value of the plant sample albumen of 2h is maximum, and the content of about 6.7ng/g, i.e. 14-3-3 albumen are most, and aluminium handles 8h, 12h
Reduction trend, respectively 4.3 ng/ μ g, 3.4 ng/ μ g, i.e. plasma membrane H is presented in the current value of plant sample albumen+- ATPase knot
There is downward trend in the 14-3-3 protein content of conjunction.
14-3-3 albumen and plasma membrane H+- ATPase, which is combined, will increase plasma membrane H+- ATPase activity, can for investigate this method
By property, tobacco is subjected to Acid-Al stress as above and is handled, plasma membrane H is measured+The activity of-ATPase.Production Phos standard first is bent
Line configures the phosphate solution within the scope of 0.05mmol/L-0.5 mmol/L with potassium dihydrogen phosphate, then by 0.25g aminobenzene
Sulfonic acid is dissolved in 1.5% Na2SO3In solution 100mL, 0.5g Na is added2SO4, sufficiently dissolve and mix, i.e., with to obtain developing solution, will show
Color liquid is added in phosphate solution, is finally the absorbance that various concentration phosphate solution is surveyed at 660nm in wavelength, and then be made
Phos standard curve y=112.4x+0.55 (y is inorganic phosphorus concentration, and x is OD value).Then, plasma membrane H is measured+The work of-ATPase
Property, the plasmalemma protein starting reaction of 500 μ g is added in 0.5mL reaction system, reaction mixture is placed in 30 DEG C of water-bath 30min
Afterwards, after reaction terminating liquid 1mL being added, developing solution 0.5mL is added, places 40min at room temperature, with spectrophotometric determination wavelength
It wherein include: 50mmol/L BTP/MES, 2mmol/L MgSO in reaction system for the light absorption value at 660-700nm4、
50mmol/L KCl、0.05% Brij(m/V)、50mmol/L KNO3、1mmol/L (NH4)6Mo7O24、1mmol/L NaN3、
5mmol/L ATP-Na2；The OD value measured is substituted into Phos standard curve, calculates the release content of Phos；1 unit
Plasma membrane H+- ATPase activity definition are as follows: under 30 DEG C of reaction condition, every milligram of proteins carry ATP decomposes release in 1min
Micromole's number of inorganic phosphate.
As a result as shown in figure 5, as aluminium handles the increase plasma membrane H of time+The activity of-ATPase presents first to rise to be declined afterwards
Trend, the plasma membrane H when aluminium handles 2h+The activity of-ATPase reaches maximum value, the electricity that this changing pattern and electrochemistry measure
Flow valuve is consistent, it was demonstrated that the reliability of this method.
14-3-3 albumen and plasma membrane H are determined by the method for co-immunoprecipitation+The combination of-ATPase, the plasma membrane of 200 μ g
2 μ g plasma membrane H are added in albumen+- ATPase antibody, 4 DEG C of incubations shake 1h (40r plasma membrane), albumin A/G of 20 μ L are then added
Plus-agarose (Santa Cruz Biotech, Santa Cruz, CA), overnight in 4 DEG C of incubation shakings.Protein sample
3500 rpm centrifugation 5min obtains protein precipitation, and the PBS (phosphate buffer) that protein precipitation is pre-chilled cleans 5 times, every time
5min；Precipitating after cleaning is dissolved with 40 μ 1 × loading of L buffer, and 40 μ L is taken to be separated by electrophoresis through SDS-PAGE (12%)
Afterwards, it is analyzed for western blotting；Protein isolate through half dry type electroporation by protein delivery to pvdf membrane, pvdf membrane
Plasma membrane H is used first+- ATPase antibody or 14-3-3 protein antibodies room temperature are incubated for 2h, then again with coupling peroxidase
The antibody room temperature of goat anti-rabbit igg is incubated for 2h, is eventually adding and generates chemiluminescent reaction substrate, is observed and is tied by gel imager
Fruit；
As a result as shown in fig. 6, A is to analyze 14-3-3 albumen and plasma membrane H using co-immunoprecipitation+The interaction water of-ATPase albumen
Flat, B, C are respectively plasma membrane H+The opposite binding capacity of-ATPase and 14-3-3 albumen, the results show that with the increasing of aluminium processing time
Add, Tobacco Root plasma membrane H+- ATPase combines the variation of 14-3-3 protein content that downward trend after first rising is presented, and at aluminium
The 14-3-3 protein content combined when managing 2h reaches maximum value, is gradually reduced later, illustrates that the aluminium processing of 50 μm of ol/L reduces tobacco
Plasma membrane H in root+The interaction of-ATPase and 14-3-3 albumen.
Through comparing, measured using the method for the present invention with plasma membrane H+- ATPase combines the changing pattern of 14-3-3 protein content
With plasma membrane H+The active variation tendency of-ATPase is consistent, also analyzes 14-3-3 albumen and plasma membrane H with co-immunoprecipitation method+-
The result of ATPase interactions between protein level is consistent.
Claims (9)
1. a kind of measurement 14-3-3 albumen and plasma membrane H+The electrochemical method of-ATPase interactions between protein, it is characterised in that including following
Step:
(1) working electrode on screen printing electrode is activated, working electrode surface is made to generate carboxyl；
(2) 14-3-3 protein antibodies liquid, 4 DEG C of combinations are added dropwise on the working electrode of the screen printing electrode after step (1) activation
After 5~20h, with electrode washing lotion flushing work electrode 3~4 times, drying；Bovine serum albumin(BSA) fluid-tight is added dropwise again and closes active site,
Room temperature closes 0.5h~2h, electrode washing lotion flushing work electrode 3~4 times, dries；
(3) 14-3- of the 5 μ L concentration within the scope of 0 μ of μ g/mL~12 g/mL is added dropwise on multiple working electrodes of step (2) drying
3 protein liquids, in conjunction with electrode washing lotion flushing work electrode after 1~2h, drying；
(4) the Biotin-14-3-3 antibody liquid of biotin labeling is added dropwise on the working electrode (s, is rushed in conjunction with electrode washing lotion after 1~2h
Working electrode is washed, is dried；
(5) horseradish peroxidase-labeled solution of streptavidin is added dropwise on the working electrode (s, reacts at room temperature 10~60min, uses
Electrode washing lotion is cleaned, and 3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions is added dropwise on screen printing electrode, then in electrification
Size of current is measured using cyclic voltammetry and current versus time curve method in credit analyzer, with 14-3-3 protein content and correspondence
Current value, make working curve, obtain equation of linear regression；
(6) plasma membrane H is added dropwise on the working electrode of the screen printing electrode after step (1) activation+- ATPase antibody liquid, as catching
Obtain probe, after 4 DEG C combine 5~20h, the same step of subsequent operation (2), working electrode made from the step is electrochemical sensing
Device；The object to be detected extracted from plant root is added dropwise on electrochemical sensor, rinses electrochemistry in conjunction with electrode washing lotion after 1~2h
Sensor, to wash away the foreign protein of non-specific binding；The Biotin-14- of biotin labeling is added dropwise on electrochemical sensor
3-3 antibody rinses electrochemical sensor, drying in conjunction with electrode washing lotion after 1~2h as signal probe；It is surveyed by step (5) method
Constant current size brings the current value measured in equation of linear regression into, obtains and plasma membrane H+The 14-3-3 egg that-ATPase is combined
White amount, and then analyze plasma membrane H+The interaction of-ATPase and 14-3-3 albumen.
2. the measurement 14-3-3 albumen and plasma membrane H according to claim 1+The electrochemical method of-ATPase interactions between protein,
Be characterized in that: the activation method of working electrode is that 80 μ of μ L~200 L are added dropwise in the working electrode surface on screen printing electrode
Electrolyte solution carries out cyclic voltammetry scan with electrochemical analyser, and working electrode surface is made to generate carboxyl；Wherein electrolyte is molten
Liquid is the phosphate buffer of 8~15mmol/L of concentration, pH 7.4；The voltage of cyclic voltammetry scan is -0.3~0.4V, sweeps speed
For 500mV/s, 10 circles are scanned altogether.
3. measurement 14-3-3 albumen according to claim 1 and plasma membrane H+The electrochemical method of-ATPase interactions between protein,
Be characterized in that: electrode washing lotion is the phosphate buffer that pH is 7.4, contains 0.05~0.2mmol/L in phosphate buffer
The Tween-20 that NaCl and mass concentration are 1%.
4. measurement 14-3-3 albumen according to claim 1 and plasma membrane H+The electrochemical method of-ATPase interactions between protein,
Be characterized in that: it is 18 μ that 14-3-3 protein antibodies liquid, which is configured to concentration with the phosphate buffered solutions containing 1~2% bovine serum albumin(BSA),
The μ g/mL of g/mL~30, dripping quantity are 3 μ of μ L -6 L.
5. measurement 14-3-3 albumen according to claim 1 and plasma membrane H+The electrochemical method of-ATPase interactions between protein,
Be characterized in that: bovine serum albumin white liquor uses the phosphate buffered saline of pH 7.4, and mass concentration is 1~2%, dripping quantity 5
~10 μ l.
6. measurement 14-3-3 albumen according to claim 1 and plasma membrane H+The electrochemical method of-ATPase interactions between protein,
Be characterized in that: the Biotin-14-3-3 antibody liquid of biotin labeling is that Biotion-XX-NHS is dissolved in N, N- dimethyl methyl
In amide, then 14-3-3 antibody is dissolved in the phosphate buffer that 0.008~0.015mol/L pH is 7.4, by above-mentioned two
After a solution mixing, 1~4h of reaction is stirred at room temperature, reaction solution crosses gel column, is with 0.008mol/L~0.015mol/L pH
7.4 phosphate buffer elution, collects eluent to get the Biotin-14-3-3 antibody of biotin labeling is arrived, wherein
The mass ratio of Biotion-XX-NHS and 14-3-3 albumen is 15-25:1.
7. measurement 14-3-3 albumen according to claim 6 and plasma membrane H+The electrochemical method of-ATPase interactions between protein,
Be characterized in that: the Biotin-14-3-3 antibody liquid concentration of biotin labeling is the 25 μ g/mL of μ g/mL~200, and 3~6 μ l are added dropwise.
8. measurement 14-3-3 albumen according to claim 1 and plasma membrane H+The electrochemical method of-ATPase interactions between protein,
Be characterized in that: horseradish peroxidase-labeled solution of streptavidin is to be dissolved in horseradish peroxidase-labeled Streptavidin
In phosphate buffered solutions containing 1~2% bovine serum albumin(BSA), concentration is the 0.5 μ g/mL of μ g/mL~3, and dripping quantity is 3~6 μ L.
9. measurement 14-3-3 albumen according to claim 1 and plasma membrane H+The electrochemical method of-ATPase interactions between protein,
It is characterized in that: plasma membrane H in step (6)+- ATPase antibody liquid concentration is 18~30 μ g/mL, and dripping quantity is 3~6 μ L.
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