GFP and fluorescence resonance energy transfer techniques for protein interaction assays
GFP and fluorescence resonance energy transfer techniques for protein interaction assays
We have divided the protocol into three phases: the first phase describes the preparation of proteins and the labeling of proteins with fluorescent dyes; the second phase, the introduction of the appropriate probe components into the cells by transfection or microinjection; and the third phase, the process of collecting and analyzing the images. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by J. Sambrook D.W. Russell.
Operation method
GFP and fluorescence resonance energy transfer techniques for protein interaction assays
Materials and Instruments
Cell lines for transfection Cells expressing the protein probes were prepared as described in stage 2 Move Phase I Labeling of proteins with fluorescent dyes For more product details, please visit Aladdin Scientific website.
N-dihydroxyethylglycine Digestion buffer N N-dimethylformamide Sodium phosphate Phosphate buffer solution TE Papain Cy3 and Cy5 OSu Monofunctional thioindolocarbocyanine succinimidyl ester Antibody SDS-Polyacrylamide gel Gelatin solution Poly-L-lysine Plasmid DNA GFP Fusion vector CO2-Undependent imaging medium
Centricon Concentrator Chromatography Columns Protein A-Agarose Gel Columns Visible Absorption Spectrophotometer Tissue Culture Plates Glass-Bottomed Tissue Petri Dishes Magnifiers Argon Laser Wideband Insulated Reflector MCP CCD GFP Cy3 Single-Sided Silver-Plated Reflector Curved Mirror Holder Lens Holder Javelin Rear Holder Inverted Microscopes Variable Aperture Lenses Mercury Lamps Oil-jointed Objective Lens Optical Bench Power PC Water Recirculation Cooling Unit
Materials
Buffers and solutions
Dilute the stock solution to the appropriate concentration.
N-dihydroxyethylglycine (0.1 mol/L, pH 7.5) adjusted with NaOH
N-Dihydroxyethylglycine adjusted with NaOH (1 mol/L, pH 9.0~9.5)
Digestive buffer
20 mmol/L sodium phosphate
10 mmol/LEDTA
20 mmol/L cysteine/HCl buffer (pH 7.0)
N,N-dimethylformamide (DMF)
Add approximately 1/3 of the volume of hygroscopic resin to the storage container for drying DMF. e.g. AG501-X8 Mixed Bed Resin (Bio-Rad) can always be placed in the DMF feedstock bottle. If there is any doubt about the integrity of the DMF, additional fresh resin can be added or the old resin can be replaced with fresh resin. This is done in anticipation of problems with the labeled proteins achieving the desired ratio, as the presence of water (which may be derived from the DMF) during the labeling reaction competes with the free dye.
Sodium phosphate (20 mmol/L) containing 10 mmol/L EDTA (pH 7.0)
Phosphate buffer solution (PBS)
TE (pH 7.0)
Enzymes and buffers
Papain immobilized on agarose pellets (50% suspension dispersed in digestion buffer; Sigma)
Labeling compounds
Cy3 and Cy5 OSu monofunctional thioindolocarbocyanine succinimidyl esters (Amersham)
Succinimidyl ester dyes of thioindolocarbocyanines (cy) are covalently bound to the free amino group (α-amino terminus of the lysine side chain or e-amino group).Cy dyes are used in lyophilized form and in all cases are kept in a dry state to avoid reaction with water. Due to possible lot-to-lot variations, the amount of dye in each bottle must be determined individually. In the example given here, Oregon green succinimidyl ester (molecular probe) was used to label the MC5 PKCα antibody.
Antibody
Purified antibodies to be labeled, such as MC5 (monoclonal) or T250 (polyclonal), or protein labeling reactions must be carried out in a buffer without amino groups. Many commercial antibody products are supplemented with free amino additives (e.g., bovine serum albumin or gelatin) that may compete with the Cy dye. There are also many suppliers who can provide antibody products without these compounds upon request, in which case a sample dissolved in phosphate buffer solution at a concentration of lmg/ml can be requested.
Gels
SDS-Polyacrylamide Gel
When preparing SDS-polyacrylamide gels to isolate proteins, refer to Appendix 8
Specialized Devices
Centricon Concentrator: YM10, YM30, YM100 (cuts molecules of 10, 30, 100 kDa size respectively; Amicon)
Gel filtration/molecular size exclusion column (pre-loaded with disposable Econo-Pac 10DG; Bio-Rad)
Protein A-agarose gel column (2 ml column from Econo-Pac; Bio-Rad)
Equilibrate the column by flushing with 20 mmol/L sodium phosphate at 20 times the volume of the charge. Protein G is recommended for purification of mouse antibodies.
UV/Visible Absorption Spectrophotometer
Additional Reagents
Step 7 in this protocol requires reagents (e.g., Bradford Analytical Reagents) for determining protein concentration (Spector et al. 1998, Chapter 56).
Methods
Fab Fragment Preparation and Purification
1. Using a disposable concentrator such as the Centricon YM100, the antibody is concentrated by centrifugation to 15-20 mg/ml in 20 mmol/L sodium phosphate solution (pH 7.0) containing 10 mmol/L EDTA.
2. Add 500ul of digestion buffer and 500ul of 50% papain immobilized on agarose particles (dispersed in digestion buffer) to 250ul of concentrated antibody product (about 4~5 mg) and digest by shaking (300~400r/min in a shaking incubator) for 6~10 hours at 37°C.
Important: Excessive digestion (e.g. more than 16 h) will result in cleavage of the Fab fragments into smaller peptides. Therefore, it is recommended to perform a pre-test by removing a small amount of the digest (~5ul) every 1h for trussing by reduced SDS polyacrylamide gel electrophoresis, where the heavy and light chains of lgG migrate like 50kDa and 25kDa peptides under reducing conditions. Papain-digested antibodies migrate as ~25kDa-sized molecules.
3. Fab fragments are purified from the Fc fraction and the undigested antibody using a protein A-agarose gel column. The digestion mixture is added to the equilibrated column and collection of the flow-through fraction is started immediately at 1 ml per portion.
Protein A will bind to the Fc portion of the antibody, so the whole antibody and the Fc portion can be removed from the digestion mixture and the resulting flow-through will contain only the Fab fragments. Many subclasses of IgG molecules do not bind to protein A. Protein G is used when Fab fragments of mouse monoclonal antibodies are to be prepared.
4. To identify which fractions of the flow-through contain purified Fab fragments, measure the OD280 of each fraction, and collect and combine the three or four fractions with the highest concentration of Fab.
Typically, Fab fragments can be found in approximately 3 to 4 fractions.
5. Concentrate the combined fractions to a volume of less than 0.5 ml by centrifugation using a Centricon YM100 column (10kDa cutoff; Amicon).
6. Add the sample to a PBS equilibrated low molecular mass (below 6kDa) gel filtration column and elute the Fab fragments with PBS (follow product instructions).
To label the Fab fragments, they must be exchanged into a buffer that does not contain free amino groups (e.g. free cysteine must be removed from the digest).
7. After elution, the protein concentration is determined by concentrating the Fab fragments using a Centricon concentrator. Add 1/10 v/v of 1mol/L N-dihydroxyethylglycine (pH 9.0)
The IgG concentration was determined by Bradford assay using bovine blood albumin as standard and multiplied by a factor of two.
The concentrated Fab product is now ready for labeling with fluorescent thioindolocyanine dye.
Dye Preparation
8. The contents of each vial of lyophilized dye are resuspended in 20ul of dry N,N-dimethylformamide to give a Cy solution of approximately 10 mmol/L. The contents of each vial of lyophilized dye are then mixed with 20ul of dry N,N-dimethylformamide.
9. 1ul of the dye mixture is diluted 1:10,000 with PBS and the concentration of the dye is calculated from the visible absorption peaks.
The absorbance coefficients of Cy3 and Cy5 at 554 nm and 650 nm are 150,000 L-nmol-1 and 250,000 L-nmol-1, respectively.
Dye binding
10. When labeling proteins, suspend them in a buffer that does not contain free amino groups. If the protein solution is not in an appropriate buffer, follow steps 6-7 above.
If the protein must be held in a buffer system for stability reasons, the gel filtration system is also equilibrated with that buffer. Note that the buffer composition should not contain free amino groups or components that inhibit labeling reactions. It is worth noting that reducing agents like dithiothreitol and β-hydrophobic ethanol interfere with the labeling reaction. However, if the addition of a reducing agent is necessary to maintain physiological/protein function, β-phobic ethanol is the preferred reducing agent because of its lower interference. Prior to labeling in 0.1 mol/L N-dihydroxyethylglycine (PH9.0), both T(P)250 and MC5 were displaced into PBS.
It is recommended that alkaline pH strips be maintained during the labeling reaction (while maintaining protein integrity) to allow the ε-amino group of lysine to increase proton dissociation and therefore bind efficiently.
11. React protein samples (antibodies, Fab fragments or proteins) with 10-40 times the excess molar amount of Cy3 for 30 min at room temperature (as described in steps 8-9). Add the dye very slowly to the solution stirred with a pipette tip (proceed carefully to avoid direct contact of the dye with the microcentrifuge tube).
To avoid denaturation of the protein by DMF, the volume of Cy3/DMF added must not exceed 10% of the total volume.
The optimal molar excess and reaction time are determined empirically. The goal of labeling is to bind 1 to 3 dye molecules per protein molecule (for a discussion of labeling ratios, see the Introduction to this protocol). The level of labeling varies greatly from protein to protein or antibody to antibody. If the final labeling ratio is too low, it may be possible to relabel the antibody. Antibodies such as T(p)250 and MC5 have been successfully labeled with more than 40-fold excess molarities of dye.
12. Terminate the labeling reaction by adding a buffer containing free amino acids, such as Tris, at a final concentration of 10 mmol/L.
13. A gel filtration column system (10DG, Bic-Rad) is used to exchange the buffer to remove excess unreacted dye and elute the egg mass into PBS.
a. Equilibrate the column system with a triple packing volume of PBS or other buffer of choice (approximately 30 ml).
b. For maximum separation, add the labeled reaction mixture directly to the resin, taking care to keep it as small as possible.
c. Wash the column with 2.5 ml of buffer (empty volume is approximately 3.3 ml) and discard the eluent.
d. Add an additional 2 ml of buffer and collect the visible protein fraction, discarding the remainder. The labeled product will run at the front and be visible to the naked eye, while the free dye is slower to elute from the column.
14. Concentrate the labeled protein again using a Centricon concentrator (which truncates molecules of appropriate size).
15. The labeled products are analyzed by SDS polyacrylamide gel electrophoresis in order to verify that the proteins were successfully labeled. After the products are separated by electrophoresis, the visible labeling products are examined directly on the gel using a UV transilluminator (302 nm).
The fluorescent band migrates to the expected mass fraction of the protein. There is no fluorescence of the free dye at the front of the electrophoretic migration;
For most dyes, UV excitation at 302nm is preferred. If no bands can be seen when irradiating the gel, a shorter wavelength of excitation light may be considered.
16. After the labeling reaction, calculate the labeling rate using the following formula:
Aλ X M / [ ( A280-ƒ X Aλ ) X ελ ]
where Aλ is the absorption of the dye at its maximum absorption wavelength λ, A280 is the absorption of the protein at 280 nm, M is the molecular mass of the protein in kDa, and ελ is the molar extinction coefficient of the dye at wavelength λ in L-mmol-1-cm-1. The formula is corrected for the absorption of the dye at 280 nm. The coefficient ƒ is the ratio of the absorbance of the dye at 280 nm to the maximum visible absorption at λ. For example, the formula for the labeling rate of a Cy3-labeled antibody becomes:
A554 X 170 / [ ( A280 - 0.05 X A554 ) X 150 ]
In order to verify whether the binding of the dye affects the physiological function of the Fab, the whole antibody, or the protein, the specific activity of the labeled product is compared to that of the corresponding unlabeled component. Specific activities include catalytic or protein binding activities. These protein binding assays can be performed using agarose-immobilized peptide ligands or phosphotyrosine particles. Standard protocols for such binding assays can be found in the Laboratory Guide to Antibody Technology (Using Antibodies) (Harlow and Lane 1999). (The Chinese translation of this book was published in September, 2002 by Science Press. ------ Editor's Note)
Cell Preparation for Phase II FLIM.FRET Analysis
Materials.
Buffers and solutions
Dilute the storage solution to the appropriate concentration.
Gelatin solution (0.1%)
Optional, see step 1.
Dissolve 0.5 g of gelatin (Porcine, Sigma) in 500 ml of 1X mash buffer solution. Autoclave.
Phosphate buffer solution (PBS)
Poly-L-lysine
Optional, see step 1.
A 0.1% (m/V) aqueous solution can be purchased from Sigma and diluted 1:10 with water prior to use.
Nucleic Acids and Oligonucleotides
Probe-containing plasmid DNA
GFP fusion vector (CLONTECH) encoding the target protein.
Culture medium
CO2-independent imaging medium
This low background fluorescent medium can be purchased from Life Technologies, or used after the following components have been removed from the standard composition of Esgle's medium as modified by Dulbecco: pH marker phenol red, penicillin, streptomycin, folic acid, and vitamin B2. The medium is supplemented with sterile HEPES (final concentration of 50 mmol/L), adjusted to pH 7.4 with NaOH, prior to use. The medium was supplemented with sterile HEPES (final concentration 50 mmol/L) adjusted to pH 7.4 with NaOH before use.
Imaging media must be free of autofluorescent components.
Specialized Devices
Tissue culture plates (6 or 12 wells) (Nunc), or
Glass-bottomed tissue culture dishes (35 mm, MatTek Corporation), or
Chamber slides or chamber coverslips (Labtek, Nunc).
Additional reagents
Step 2 in this protocol requires the reagents needed for the transfection method described in Chapter 16.
Cells and Tissues
Cell lines for transfection (adherent or suspension culture)
In addition to considering the suitability of the cell line for the system under study, the only other consideration in the selection of cell types is the ease with which they can be immobilized by a number of methods (see step 1 for instructions).
Methods
1. Transplant the cells onto a suitable surface for microscopic observation:
For live cell products: spread the cells onto a glass-bottomed flat dish or chamber coverslip.
For post-transfected fixed cell products: transplant cells onto a glass coverslip placed in a 6- or 12-well tissue culture dish.
The final choice of vessel depends on whether the fine holders require microinjection after transfection; if so . The shape and remaining space of the vessel should be considered in light of the need for needle manipulation.
For suspension cells, curettes are used to facilitate the use of media like gelatin (0.1%, m/V, PBS; autoclaved) or poly-L-lysine (0.01% aqueous solution, m/V). In both cases, the selected medium was used to cover the surface of the wells, coverslips or coverslips for about 30 min to coat the surface of the above materials, and then the excess medium was aspirated out, and then the cells were transplanted directly onto the surface covered by the medium. The cells can also be kept in suspension during the transfection operation, and then cured immediately before imaging. Once cured, the cells are ready for microinjection.
The cells are applied to the plate at a -determined density so that approximately 40% of the cells can be fused the next day.
2. Transfect cells with a plasmid encoding a GFP-tagged target protein using any of the transfection methods described in Chapter 16.
3. Incubate the transfected cells under appropriate conditions for 16~24 h to allow the cells to express the target protein.
In some cases, the level of protein expression must be precisely controlled. In this case, it is recommended to optimize the transfection efficiency and the expression cycle before performing the FLIM assay, either by using adjusted enhancers to promote protein expression (see Chapter 17) or by using nuclear microinjection as an alternative to transfection (see "Alternatives, Microinjection of Live Cells" at the end of this protocol).
4. Identification of cells expressing the target protein.
5. (Optional) If the experimental design allows, choose one of the alternatives following step 6, i.e., microinjection (for live cells) or fixation and staining (for fixed cells), to introduce another probe (e.g., labeled protein).
6. Immediately prior to microscopy, replace the medium with CO2 non-dependent imaging medium (commercially available medium or Dulbecco's modified Eagle's medium adjusted as described in the Materials section).
In the experimental design, if serum starvation of the cells is required prior to the treatment in question, the cells are starved for the time period described prior to imaging. However, if apoptosis occurs in the absence of plasma, the cells should be placed in 0.5% fetal bovine blood淸 during the starvation period and then replaced with blood-free medium immediately prior to imaging. 
Phase III FLIM-FRET measurement
MATERIALS
Specialized device
Amplifier (ENI403LA or Intra-actionPA-4)
Argon laser (Coherent, Innova70C)
Broadband insulated mirrors (2; Newport Corporation)
Detector
MCP, HamamatsuC5825 or LaVision Picostat HR
CCD, Photometries Quamix with Kodak KAF 1400 chip
Filter
GFP,OG(Q495LP,HQ5lO/20;Chroma)
Cy3(HQ545/30,Q565LP,HQ610/75;Chroma)
Single-sided silver-plated reflector (Zeiss)
Curved lens holders (2), lens mounts (2), standard poles (5), and rear mounts (5) (Newport)
Inverted Microscope (Zeiss 135TV)
IPLab Spectrum (Signal Analytics)
Variable Aperture (Comar)
Lenses (focal lengths 12 and 7.6 cm, focal lengths 2.5 and 3.8 cm respectively; Newport)
Mercury lamp (100 W Zeiss; HBO 100 AttoArc)
Multimode fiber step-indexed 1-mm core
(Newport)
Oil-jointed objective (100X/1.4NA;ZeissFluar)
Optical bench (2X1m;TMC)
Power meter and head (Ophir, Nova Display and 2A-SH)
Power PC (Macintosh) with PCI-GPIB card (National Instruments)
Shutter (high speed) Vincent Associates, UniblitzVS25)
Shutter starter (Vincent Associates, UniblitzVS25)
Standing wave acousto-optic modulator (AOM)(Intra-action, 80MHz)
Frequency Doubling Synthesizer (IFR2023)
Variable Density Filter Pulleys (Laser Components)
Water Recirculation Cooler (Grant)
Cells and Tissues
Cells (live or immobilized) expressing the protein probe were prepared as described in Phase II
Methods
1. Adjust the wavelength-selective prism to select an argon laser at 488 nm as the excitation wavelength. The position of the highly reflective mirror surface was micro-adjusted using the control knob on the back of the laser to optimize the output power.
2. Set the frequency synthesizer to start the AOM to a resonant frequency of approximately 40MHz (for the experiments presented here, a start frequency of 40.112MHz was used). This causes the laser beam to vibrate at twice the intensity of the drive frequency (80.224MHz).
3. The diffraction as well as the modulation intensity of the AOM was optimized by adjusting the angle of incidence and monitoring the intensity of the non-diffracted zero-level light with a photometer. The output power of the zero-level light is minimized at the optimized diffraction angle (to reach the corresponding maximum diffraction).
4. turn on the MCP and CCD. set the base value of the photocathode voltage to -2V and adjust the gain to match the full power range of the CCD. This depends on the fluorescence intensity of the sample and needs to be determined empirically. Ideally, the gain should be as small as possible to minimize noise. Typically, the gain is set to 1 for the Hamamatsu C5825 and 3 for the Photometrics Quantix CCD, and the CCD readout is set to a 2X2 raster.
5. Set the main frequency synthesizer to drive the MCP at twice the frequency of the driving AOM (80.224MHz for the example above).
6. Select the most appropriate objective lens for the experiment. In this example, a Zeiss Fluar 100x/1.4NA oil objective was used.
7. In order to obtain zero-level lifetime reference images, 16 phase-dependent images are recorded at 22.5° intervals throughout the week, starting with a strong scan (e.g., a small piece of aluminum foil is placed on the imaging surface of a coverslip or glass-bottomed dish).
a. Replace the fluorescent filter component with a single-sided silver-plated reflector and minimize the intensity of the extraneous light with a variable intensity filtering pulley. Adjust the focusing point to the surface of the aluminum foil.
Note: When setting up the aluminum foil for imaging, always be careful not to look directly into the microscope with the naked eye until the intensity of the external light source has been set to a minimum.
b. Take an image of the aluminum foil with an exposure time of approximately 100ms. This image is used to select the ROI and estimate the optimum exposure time required. Because the phase of the main frequency synthesizer may not be maximal at this point, an exposure time that generates about 1000 counts should be chosen to avoid saturation of the detector.
c. Record 16 phase images at 22.5° intervals throughout the week, determine the phase in the image series when maximum intensity is reached, and reset that phase to 0 degrees on the main frequency synthesizer.
d. Record another week of sixteen phase images of the aluminum foil and note them as the zero-level life reference image.
When performing lifetime imaging, we recommend monitoring phase stability at all times. Our unit can be phase stabilized to within 0.3° over the course of an hour. For our unit, a sequence of reference foil images is recorded and saved every hour.
8. Save the external excitation light source to maximum (using the variable intensity filter pulley); this way the system is ready to acquire image data of the cells.
9. Acquire a donor image using the GFP filter setup (beam splitter: Q495LP, excitation unit: HQ510/20). The exposure time is set to achieve 75% of the CCD dynamic range (3000 counts for a 12-bit CCD). Select the region of interest in the image to determine the fluorescence lifetime.
10. Prepare a donor fluorescence lifetime map:
a. Acquire a series of 16 phase-dependent images (spaced 45° apart in phase) of two neighboring, one orthophase and the other anti-phase loops to correct for phase quenching. Optimize the exposure time setting for each phase image as in step 9.
b. Acquire another image without sample exposure. Extract this background-removed image from all phase images in the series, and then correct for donor quenching by one-level summation of each pair of images of the same phase in the forward and inverse phase loops.
c. From this information, as well as the zero-level lifetime reference image (aluminum foil), calculate the donor fluorescence lifetime profile as described in the discussion of image manipulation in the introductory section of this protocol.
11. Record the image of the acceptor and then change the illumination source to a 100W mercury lamp (Zeiss Attoarc) to quench the acceptor. Move the Cy3 filter components (exciter: HQ545/30; beam splitter: Q565LP; emitter: HQ610/75chroma) into the detection pathway. After optimizing the exposure time to fill the entire dynamic range of the CCD, acquire the receptor image. Under conditions where Cy3 fluorescence cannot be detected, the detector component is turned off and the receptor is irradiated.
Importantly, when performing photobleaching experiments with acceptors, it is extremely important to make it clear that the photoproducts of the acceptor in the donor channel do not fluoresce after photobleaching. 
