This method can help answer the questions in protein-protein interaction, will allow us to directly detect two protein located within 10 nanometer from each other inside living cell. The main advantage of this technique is that it is simple and easy to reproduce. It also requires simple materials that's available in many modern laboratories.
This measure can be used with any living tissue or culture cell that transitionally spread at protein of interest. If you use this technique for the first time, it's recommend that you should use the positive control and negative control to test the system first. To begin, sow Nicotiana benthamiana seeds in a pot containing wet soil at a high density.
Keep the planted seeds in a growth chamber set at 23 degrees Celsius. When the diameter of the euphyll reaches 0.3 to 0.5 centimeters, transfer the seedlings to larger pots and allow them to grow in the same chamber with the same parameters. Next, prepare bacterial cells for agroinfiltration by inoculating each agrobacterium colony containing the FRET constructs in five milliliters of LB medium supplemented with antibiotics and 150 micromolar acetosyringone.
Incubate the culture overnight at 28 degrees Celsius. After incubation, centrifuge the cells at 3, 000 G for five minutes at room temperature. Resuspend the cells in agroinfiltration buffer to an OD 600 of 0.5.
For double construct agroinfiltration, combine the resuspended cells at a one-to-one volume ratio with cells harboring the appropriate constructs. Incubate the cells at 28 degrees Celsius for 30 minutes to one hour. To perform agroinfiltration, load the bacterial culture into a one milliliter needleless syringe.
Gently but firmly, press the nozzle of the syringe against the abaxial side of the fully expanded Nicotiana benthamiana leaves while holding the leaf with a gloved finger on the adaxial side. Infiltrate up to four spots on a leaf, three leaves per plant, and two or three plants for each bacterial culture. Maintain the agroinfiltrated plants in the same growth chamber under the same conditions as described earlier for 24 to 36 hours.
After 24 to 36 hours of infiltration, use a razor blade to cut each agroinfiltrated leaf into small pieces of two by four millimeters between the veins. Place the leaf pieces on a glass slide with the abaxial leaf surface facing up. Place a drop of water on the leaf pieces, and cover them with the cover glass.
Slightly tap the cover glass to remove air bubbles. Then turn on the microscope and laser. Place the slide into the microscope stage holder.
To set up the parameters for SE-FRET, open the multi-dimensional acquisition tool. Then set the donor channel for excitation and emission of the donor fluorochrome GFP at an excitation laser of 405 nanometers, and emission filter at 400 to 597 nanometers. Set the acceptor channel for excitation and emission of the acceptor fluorochrome mRFP and an excitation laser of 561 nanometers and emission filter at 400 to 597 nanometers.
Set the FRET channel for excitation of the donor and emission of the acceptor fluorochromes with a 405 nanometer excitation laser and 597 to 617 nanometer emission filter. Set the donor excitation intensity at a minimum level to observe FRET while avoiding photobleaching. Excite the donor and scan for cells containing the expected fluorescence signal of the acceptor.
Select the region that contains the fluorescence signal of interest. Acquire an SE-FRET image sequence by pressing the snap button. To set up parameters for AB-FRET, utilize the donor and acceptor channel parameters set for SE-FRET, but turn off the FRET channel.
Then prepare to set the parameters for photobleaching of the acceptor mRFP. Ensure that bleaching starts after five images. Allow 200 iterations for each area bleach.
Keep 100%laser intensity at 561 nanometers. Maintain a bleaching duration of 45 seconds. Ensure a scan speed of 512 by 512 pixels at 400 hertz.
Search and draw the region of the cell to be bleached, and activate bleaching by pressing the start experiment button. In case of the positive control, after five images, the bleaching starts, and the region of interest turns green. Check mean ROI to confirm that the intensity of mRFP is decreasing while that of GFP is increasing.
Alternatively, check layout, which shows that mRFP was bleaching and GFP was increasing. To analyze SE-FRET data, use the ImageJ software. Open the images and create a stack of donor only.
Save the images in eight bit as TIF file. Similarly, create the stack for acceptor only, then create a combined stack of donor and acceptor. Next, use the PixFRET plugin to generate images of the SE-FRET efficiency.
Open the created stacks, and generate the corrected FRET images after subtracting the spectral bleed through. Present the image as pseudocolor image. To analyze the AB-FRET data, calculate percent AB-FRET as the percent increase in GFP emission after mRFP photobleaching using this formula.
Specific protein-protein interaction of the histone deubiquitinase OTLD1 with a transcription factor LSH10 was studied by SE-FRET. The cell nuclei were simultaneously recorded in three channels, and a pseudocolor scale was generated to depict the SE-FRET efficiency. The transition from blue to red corresponds to an increase in FRET efficiency and protein-protein proximity.
SE-FRET intensity following the co-expression of LSH10 and OTLD1 was comparable to that observed for the positive control mRFP-GFP. No SE-FRET was observed in negative controls such as co-expression of OTLD1, mRFP, and LSH4-GFP, or free mRFP and LSH10-GFP. AB-FRET images showed that the co-expression of LSH10-GFP and OTLD1-mRFP resulted in an increased GFP donor fluorescence after the RFP acceptor was photobleached.
A similar increase in donor fluorescence was observed in the positive control, but not in the negative controls when the acceptor fluorescence was inactivated. Quantitative analysis of the AB-FRET data demonstrated a statistically significant increase in donor fluorescence after coexpressing LSH10 and OTLD1. The positive control produced a percent AB-FRET of approximately 30%whereas the negative controls produced none.
SE-FRET and AB-FRET showed a signal in the cell nucleus consistent with the subcellular localization of the transcription factor histone modifying enzyme complexes as well as the nucleocytoplasmic nature of the GFP-MRFP proteins. The most important thing to remember before following the protocol is to test into the right donor acceptor express pair depending on the confocal nanoscopy model. After running and doing our iteration, it's only 24 to 36 hour to receive the results.
After watching this video, one should have a good understanding of how to use FRET technique to study protein-protein interaction.
