CN104165873A - Method for detecting co-localization degree of two membrane proteins in live plant cell - Google Patents
Method for detecting co-localization degree of two membrane proteins in live plant cell Download PDFInfo
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Abstract
The invention provides a method for detecting the co-localization degree of two membrane proteins in a live plant cell. The method comprises the following steps (1) co-expressing two fusion proteins in a plant so as to obtain a plant material labeled in two colors by two proteins: namely a fluorescent protein A labeled protein A to be measured, a fluorescent protein B labeled protein B to be measured, wherein the fluorescent protein A and the fluorescent protein B give off different fluorescence; (2) carrying out observation on the plant material by using an angle-variable total internal reflection fluorescent microscope so as to obtain a two-channel image; (3) utilizing Image J software to convert the obtained two-channel image into a lossless compression TIFF mode, calibrating two images through PPA software, then removing the background in the images by utilizing median filter, and finally using dual Gauss fitting to calculate the approach index between the protein A and the protein B so as to obtain the co-localization degree of the protein A and the protein B. The method has the advantages of simpleness and good repeatability, avoids the interference of fluorescent signals in the cell, and obtains values having definite biological meanings.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method that detects in live plant cell two kinds of memebrane proteins and locate altogether degree.
Background technology
As the Primary Actor of cytoactive and function, protein has played irreplaceable effect in many physiology courses.The regulation and control that protein participates in DNA replication dna, transcribes mainly with the form of homology or allos compound, the regulation and control of gene expression, in the vital movement such as signal transduction and metabolic process of synthetic and secretion, the cell of protein.Therefore, the interaction between Study on Protein molecule is significant.
It is a kind of index of weighing common location degree that albumen approaches index (PPI, protein proximity index).By comparing two respectively with the fluorescence signal similarity degree of fluorescently-labeled protein, the degree of these the two kinds of protein interactions of the higher explanation of PPI value that obtain is higher.With Pearson correlation coefficient (Pearson ' s correlation coefficient), altogether compared with other similar method such as orientation factor (Colocalization coefficients), PPI value is more accurate, and there is clearer and more definite biological significance, under the interference that there is no non-specific fluorescence, the numerical value of PPI is equivalent to the number percent of common localized molecules.In addition, it can also rejection image heterogeneity on assessing the impact of common location degree.
Variable angle utilizing total internal reflection fluorescence microscope (VA-TIRFM, variable angle-total internal reflection fluorescence microscopy) evanescent wave (evanescent field) excited sample of utilizing total internal reflection to produce, field of illumination is limited to the thin layer of sample surfaces 100nm left and right.The fluorophor that is only positioned at this thin layer in sample just can be excited, thereby has avoided the interference of sample interior fluorophor to imaging.VA-TIRFM imaging device is simple and easily combine with other imaging and Detection Techniques, and the imaging effect of high s/n ratio can be provided, and high resolving power on z direction of principal axis.
Summary of the invention
The object of this invention is to provide a kind of method that detects in live plant cell two kinds of memebrane proteins and locate altogether degree, comprise the steps:
(1) make the following two kinds of fusions of coexpression in plant, to obtain the vegetable material that two kinds of albumen to be measured has been carried out to Bicolor-code: the testing protein A of fluorescin A mark, the testing protein B of fluorescent protein B mark, fluorescin A and fluorescent protein B are sent out the fluorescence of different colours;
(2) treat measuring plants material with variable angle utilizing total internal reflection fluorescence microscope and observe, obtain Channel Image;
(3) with ImageJ software, gained Channel Image is saved as to the tiff format of Lossless Compression, then in PPA software, two images are calibrated, with medium filtering, image is gone to background process again, finally by the index that approaches of double gauss the Fitting Calculation testing protein A and testing protein B, thereby obtain the common location degree of testing protein A and testing protein B.
Described in above-mentioned steps (1), fluorescin A and fluorescent protein B are green fluorescent protein GFP or red fluorescent protein mCherry.
Testing protein A and testing protein B described in above-mentioned steps (1) are that arabidopsis is to light element phot1 or clathrin light-chain clathrin light chain CLC.
Described in above-mentioned steps (1), plant is arabidopsis.
The image producing when the image that Channel Image described in above-mentioned steps (2) produces while referring to the testing protein A that detects fluorescin A mark and the testing protein B that detects fluorescent protein B mark.
Described in above-mentioned steps (2), in observation process, the excitation wavelength that detects the testing protein use of Green Fluorescent Protein is 473nm or 488nm, and the excitation wavelength that detects the testing protein use of red fluorescent protein mark is 543nm or 561nm.
Described in above-mentioned steps (2), in observation process, the time shutter is 100ms or 200ms;
And/or vegetable material to be measured is live plant material described in step (2), described live plant material is specially the root of plant seedlings plumular axis or plant seedlings or the blade of plant seedlings.
Described in above-mentioned steps (2) in observation process, microscopic examination to plant tissue be plant roots epidermal cell or plumular axis epidermal cell or blade lower epidermis cell.
The method that with ImageJ software, gained Channel Image is saved as to the tiff format of Lossless Compression described in above-mentioned steps (3) is: in ImageJ software, open described Channel Image, by Image->Type, image is converted into 16-bit, click Edit->Options->Input/Output ... it is 100 that JPEG Quality (1-100) is set, and then two images is all saved as to tiff format.
The method of in PPA software, two images being calibrated described in above-mentioned steps (3) is: in PPA software, open the tiff format image of described Lossless Compression, click Show->Images and determine two numbering Image1 and Image2 that image is corresponding; After operation Analyze->Alignment, the information of returning will have two kinds; If return message is " No shift needed ", image does not need to calibrate, and clicks " OK " and can complete image calibration; If return message is " Max correlation is at (X, Y), shift the image pair? ", to judge whether to calibrate according to the value of X and Y, in the time that the absolute value of X and Y is all less than 50, proofread and correct, click "Yes"; In the time that the absolute value of X and Y is greater than 50, two image focal planes may not overlap, and are not suitable for carrying out the calculating of common location degree, need to change one group of image this time and calculate.
Described in above-mentioned steps (3), go the method for background process to be with medium filtering to image: in PPA software, operation Analyze->Median Filter, determines that in the dialog box ejecting, two options are all selected; " Large Square Size " is set to 16 or 32, and " Small Square Size " is set to 2 or 3; Click " OK ", complete background process.
The method of the common location degree by double gauss the Fitting Calculation testing protein A and testing protein B described in above-mentioned steps (3) is: in PPA software, operation Analyze->Correlation->Cross-correlation, in the interface of ejecting, clicking " Contour " makes simple crosscorrelation curve show with the form of isogram, click again " Enter Draw Mode ", in isogram, draw one by the straight line of initial point along slow peak, the right button of clicking the mouse, selects " Shift to zero ".Then choose " Autocorrelation1 ", click " Fit "; Choose again " Autocorrelation2 ", click " Fit "; Finally choose " Crosscorrelation ", click " Fit ", the numerical value PPI1 that double gauss matching is returned and PPI2 are respectively Image1 and the corresponding albumen of Image2 approaches exponential quantity; Click File->Add Current Result, then move Show->Result, can see all parameters and result.
PPA software described in above-mentioned steps (3) is PPA (protein proximity analyzer) software.
Software used in said method all can obtain light version on the internet, and ImageJ software download address is http://rsb.info.nih.gov/ij/, and PPA software download address is http://www.anes.ucla.edu/～wuyong/.
The invention provides a kind of method that detects in live plant cell two kinds of memebrane proteins and locate altogether degree.Experimental result shows: have 40.7% phot1 and CLC to locate altogether, have 36.4% CLC and phot1 to locate altogether.It is simple, reproducible that the present invention obtains the method that two kinds of memebrane proteins locate degree altogether, avoided the interference of cell interior fluorescence signal, and the numerical value obtaining has clear and definite biological significance, for further Way for Studying Protein-Protein Interactions provides favorable method.
Brief description of the drawings
Fig. 1 is phot1-GFP (left side) and CLC-mCherry (right side) image that variable angle utilizing total internal reflection fluorescence microscope photographs.
Fig. 2 be phot1-GFP (left side), CLC-mCherry later of format conversion, image calibration and background removal (in) and the superpose image on (right side) of two passages.
Fig. 3 is the simple crosscorrelation isogram of having drawn through initial point straight line.
Fig. 4 is the three-dimensional plot of phot1-GFP and CLC-mCherry simple crosscorrelation degree.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
In embodiment 1, quantitative test arabidopsis, locate altogether degree to light element phot1 and clathrin clathrin
For detection of two kinds of albumen of common location degree be respectively arabidopsis to light element phot1 and clathrin light-chain clathrin light chain CLC, these two kinds of albumen all have expression on the epidermal cell film of arabidopsis plumular axis.Green fluorescent protein GFP mark phot1, red fluorescent protein mCherry mark CLC.
One, vegetable material
1, the acquisition of transgenic arabidopsis
The arabidopsis of what this experiment was used proceed to fusion phot1-GFP and CLC-mCherry disclosed in document " Wan; et al.Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana.Plant Methods; 2011; 7:27 ", and the public can obtain from Beijing Forestry University.
2, the cultivation of arabidopsis seed
The arabidopsis seed with Two Colour Fluorescence protein labeling is placed in to 1.5ml centrifuge tube, add 2.5% sodium hypochlorite to soak 10min, clean 5 times with aqua sterilisa subsequently, be seeded into and contain on the 1/2MS nutrient culture media that 1.0% sucrose, 1.0% agar, pH value are 5.8, cultivate 4 days, obtain Arabidopsis thaliana Seedlings for 22 DEG C.Arabidopsis thaliana Seedlings plumular axis is test material.
Two, locate altogether degree detecting
(1) variable angle utilizing total internal reflection fluorescence microscope observing samples obtain image
Above-mentioned Arabidopsis thaliana Seedlings plumular axis is observed with variable angle utilizing total internal reflection fluorescence microscope, time shutter is 200ms, green channel excitation wavelength is 473nm, red channel excitation wavelength is 561nm, EMCCD yield value is 345, Arabidopsis thaliana Seedlings plumular axis under the same visual field is carried out to binary channels shooting, obtain Channel Image.Fig. 1 is with the green channel phot1-GFP (left side) of variable angle utilizing total internal reflection fluorescence microscope shooting and the original image of red channel CLC-mCherry (right side).
(2) image format conversion
(1) start ImageJ (version is 1.42q) software, first set Image Saving quality.Click Edit->Options->Input/Output ... it is 100 that JPEG Quality (1-100) is set, option " Save TIFF and Raw in Intel Byte Order " is chosen, other option is default value, clicks " OK ".
(2) in ImageJ software, open above-mentioned gained green channel image (File->Open-> selects picture-> to open), select Image->Type->16-bit, then picture is saved as to tiff format (File->Save As->Tiff), image name is phot1-GFP.GIF.Above-mentioned gained red channel image is also carried out to same operation, obtain the tiff format image of Lossless Compression, image name is CLC-mCherry.GIF, and the green channel tiff image of itself and gained Lossless Compression is kept in same file folder.
(3) image calibration and background removal
(1) start PPA software, by File->Open, choose the tiff image of two passage Lossless Compressions of above-mentioned gained simultaneously, click " opening ".Select Show->Images, in the interface of ejecting, can see the image of importing, determine two numbering Image1 and Image2 that image is corresponding.Operation Analyze->Alignment, return message is " No shift needed ", clicks " OK ".
(2) operation Analyze->Median Filter, determine that in the dialog box ejecting, two options are all selected, it is 16 that " Large Square Size " value is set, and " Small Square Size " value is 3, click " OK ", complete background process.Fig. 2 be the phot1-GFP (left side), the CLC-mCherry that cross through format conversion, image calibration and background removal (in) and the image of their stacks (right side).
(4) locate altogether the calculating of degree
(1) in PPA software, operation Analyze->Correlation->Cross-correlation.In " Line Scan Analysis " interface, click " Contour ", then select " Enter Draw Mode ", in isogram, draw straight line along slow peak, and make straight-line pass initial point.Hit right mouse button at Points on Straight Line, select " Shift to zero ".Fig. 3 is simple crosscorrelation isogram, has drawn the straight line through initial point in figure.
(2) choose " Autocorrelation1 ", click " Fit "; Choose again " Autocorrelation2 ", click " Fit "; Finally choose " Crosscorrelation ", click " Fit ".The PPI value of returning is respectively phot1 → CLC, 0.407; CLC → phot1,0.364.Result shows have 40.7% phot1 and CLC to locate altogether, has 36.4% CLC and phot1 to locate altogether.Fig. 4 is three-dimensional plot corresponding to simple crosscorrelation degree.
Method (such as FRET (fluorescence resonance energy transfer), bimolecular fluorescence complementary etc.) that method of the present invention is located degree than other altogether based on fluorescin and cell biology imaging analysis is more simple, quick, accurately, and can be quantitative.
Claims (10)
1. detect in live plant cell two kinds of memebrane proteins and locate altogether a method for degree, comprise the steps:
(1) make the following two kinds of fusions of coexpression in plant, to obtain the vegetable material that two kinds of albumen to be measured has been carried out to Bicolor-code: the testing protein A of fluorescin A mark, the testing protein B of fluorescent protein B mark, fluorescin A and fluorescent protein B are sent out the fluorescence of different colours;
(2) treat measuring plants material with variable angle utilizing total internal reflection fluorescence microscope and observe, obtain Channel Image;
(3) with ImageJ software, gained Channel Image is saved as to the tiff format of Lossless Compression, then in PPA software, two images are calibrated, with medium filtering, image is gone to background process again, finally by the index that approaches of double gauss the Fitting Calculation testing protein A and testing protein B, thereby obtain the common location degree of testing protein A and testing protein B.
2. according to the method described in right 1, it is characterized in that, in step (1), described fluorescin A and fluorescent protein B are green fluorescent protein GFP or red fluorescent protein mCherry.
3. according to the method described in right 1 or 2, it is characterized in that, in step (1), described testing protein A and testing protein B are that arabidopsis is to light element phot1 or clathrin light-chain clathrin light chain CLC.
4. according to the arbitrary described method of right 1-3, it is characterized in that, in step (1), described plant is arabidopsis.
5. according to the arbitrary described method of right 1-4, it is characterized in that, in step (2), in described observation process, the excitation wavelength that detects the testing protein use of Green Fluorescent Protein is 473nm or 488nm, and the excitation wavelength that detects the testing protein use of red fluorescent protein mark is 543nm or 561nm.
6. according to the arbitrary described method of right 1-4, it is characterized in that, in step (2), in described observation process, the time shutter is 100ms or 200ms;
And/or in step (2), described vegetable material to be measured is live plant material, described live plant material is specially the root of plant seedlings plumular axis or plant seedlings or the blade of plant seedlings.
7. according to the arbitrary described method of right 1-6, it is characterized in that, in described step (3), the method that with ImageJ software, gained Channel Image is saved as to the tiff format of Lossless Compression is: in ImageJ software, open described Channel Image, by Image->Type, image is converted into 16-bit, click Edit->Options->Input/Output ... it is 100 that JPEG Quality (1-100) is set, and then two images is all saved as to tiff format.
8. according to the arbitrary described method of right 1-7, it is characterized in that, in described step (3), the method of in PPA software, two images being calibrated is: in PPA software, open the tiff format image of described Lossless Compression, click Show->Images and determine two numbering Image1 and Image2 that image is corresponding; After operation Analyze->Alignment, the information of returning will have two kinds; If return message is " No shift needed ", image does not need to calibrate, and clicks " OK " and can complete image calibration; If return message is " Max correlation is at (X, Y), shift the image pair? ", to judge whether to calibrate according to the value of X and Y, in the time that the absolute value of X and Y is all less than 50, proofread and correct, click "Yes"; In the time that the absolute value of X and Y is greater than 50, two image focal planes may not overlap, and are not suitable for carrying out the calculating of common location degree, need to change one group of image this time and calculate.
9. according to the arbitrary described method of right 1-8, it is characterized in that, in step (3), describedly go the method for background process to be to image with medium filtering: in PPA software, operation Analyze->Median Filter, determines that in the dialog box ejecting, two options are all selected; " Large Square Size " is set to 16 or 32, and " Small Square Size " is set to 2 or 3; Click " OK ", complete background process.
10. according to the arbitrary described method of right 1-9, it is characterized in that, in step (3), the method for the described common location degree by double gauss the Fitting Calculation testing protein A and testing protein B is:
In PPA software, operation Analyze->Correlation->Cross-correlation, in the interface of ejecting, clicking " Contour " makes simple crosscorrelation curve show with the form of isogram, click again " Enter Draw Mode ", in isogram, draw one by the straight line of initial point along slow peak, the right button of clicking the mouse, selects " Shift to zero ".Then choose " Autocorrelation1 ", click " Fit "; Choose again " Autocorrelation2 ", click " Fit "; Finally choose " Crosscorrelation ", click " Fit ", the numerical value PPI1 that double gauss matching is returned and PPI2 are respectively Image1 and the corresponding albumen of Image2 approaches exponential quantity; Click File->Add Current Result, then move Show->Result, can see all parameters and result.
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| CN108195801A (en) * | 2017-11-17 | 2018-06-22 | 北京林业大学 | Single molecules level observation Stomacal guard cell memebrane protein distribution and dynamic method |
| CN112662703A (en) * | 2020-12-29 | 2021-04-16 | 北京林业大学 | Method for observing plant cell nucleus protein dynamics by using fluorescence bleaching recovery technology |
| CN112683867A (en) * | 2020-12-21 | 2021-04-20 | 复旦大学附属中山医院 | Method for detecting depigmentin D on living cells in real time and application thereof |
| WO2021159479A1 (en) * | 2020-02-14 | 2021-08-19 | 深圳华大智造科技股份有限公司 | Method for analyzing droplets on basis of image, computer device and storage medium |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021159479A1 (en) * | 2020-02-14 | 2021-08-19 | 深圳华大智造科技股份有限公司 | Method for analyzing droplets on basis of image, computer device and storage medium |
| CN112683867A (en) * | 2020-12-21 | 2021-04-20 | 复旦大学附属中山医院 | Method for detecting depigmentin D on living cells in real time and application thereof |
| CN112683867B (en) * | 2020-12-21 | 2023-08-04 | 复旦大学附属中山医院 | Method for detecting mesothelin D on living cells in real time and application thereof |
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Inventor after: Lin Jinxing Inventor after: Xue Diequn Inventor after: Zhang Liang Inventor before: Lin Jinxing Inventor before: Xue Diequn |
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Application publication date: 20141126 |