AirID is valuable enzyme for protein-protein interaction, and it creates less toxicity and less error-ness in time-taken processes than other proximity-labeling enzymes. A step-by-step protocol is presented for performing the proximity-labeling experiment in cucumber or Cucumis sativus using AT4G18020 APRR2-AirID protein as a model. Demonstrating the procedure will be Ahmad Zada, Imran Khan, Yuting Cheng and Min Zhang from my laboratory.
Begin with transferring the Cucumis sativus or cucumber seeds in water, incubating them at 50 degrees Celsius for 20 minutes, and then placing the seeds on the filter paper on a Petri plate for 12 to 16 hours. Later, transfer the seeds to pots containing soil and grow them in a climatic chamber at 23 degrees Celsius in 16 hours light and 18 hours dark photo period for three to four weeks. Transfer 2.5 microliters of the plasmid EarleyGate 100 to the competent cells of the agrobacterium tumefaciens GV3101 strain.
Incubate the tube on ice for 30 minutes before transferring it to liquid nitrogen for three minutes. To give the heat shock, transfer the tube to the incubator at 37 degrees Celsius for five minutes. Then place the tube on ice for two minutes.
Add 1, 000 microliters of Luria Bertani or LB medium to the heat shocked agrobacterium cells. After incubating the cells at 30 degrees Celsius and 118 RPM for one hour, centrifuge them at 3000 G for two minutes at four degrees Celsius. Then discard the 800 microliter supernatant and mix the remaining solution.
Plate it on the LB medium supplemented with 50 micrograms per milliliter of kanamycin and gentamycin each and 25 micrograms per milliliter of rifampicin. Incubate the plates at 30 degrees Celsius for 48 hours. Pick up some bacterial colonies from the plates.
Put them in LB liquid media, supplemented with appropriate antibiotics and incubate liquid LB at 30 degrees Celsius and 218 RPM for 36 to 48 hours. After two minutes of centrifugation at 3, 000 G at four degrees Celsius, resuspend the pellet in an agroinfiltration buffer to adjust the optical density to one at the 600 nanometer wavelength. Using a one milliliter needleless syringe, infiltrate the entire epidermis of the abaxial surface with 1.5 milliliters of the resuspended agrobacterial inoculum.
Maintain the plants in the climate chamber at 23 degrees Celsius with 75 micromoles per meter square per second light for 16 hours in dark conditions for eight hours After 36 hours, infiltrate one milliliter of five micrograms of biotin into the already infiltered leaves. After four to 12 hours of biotin infiltration, cut the leaves and quickly transfer them to liquid nitrogen to avoid protein degradation. Grind the leaves using a pestle and mortar and quickly add two milliliters of PBS BSA of pH 7.4, followed by slow grinding.
Transfer the sample mix to a 15 milliliter conical tube through a quick filtration material filter placed on top of it and keep the tube on ice. Transfer the samples to a two milliliter tube. Add a 1%protein inhibitor cocktail and mix the contents by turning the tube up and down seven to eight times before centrifuging it.
Once done, transfer the supernatant to a new two milliliter tube and add 10%beta-D-maltoside. Place the tube on ice for five minutes before centrifuging at 20, 000 G for 10 minutes at four degrees Celsius. To equilibrate the desalting column, mark one side of the column and centrifuge it with the marked side facing outward.
Remove the top and bottom cover of the column and centrifuge again at 1, 000 G for two minutes at four degrees Celsius. Discard the liquid solution from the collection tube of the column and wash the tube with five milliliters of PBS buffer with centrifugation as previously demonstrated, at least five times. Add one milliliter of PBS BSA to 50 microliters of Streptavidin-C1-conjugated magnetic beads in a 1.5 milliliter tube and mix it thoroughly.
Place the tube on a magnetic stand for three minutes to absorb the beads toward one side of the tube. Discard the supernatant from the other side and repeat this PBS BSA wash thrice. To enrich the biotinylated protein, add two milliliters of the samples to the column.
After centrifugation of the column at 1000 G for eight minutes at four degrees Celsius, add one milliliter of desalted protein extract to 50 microliters of Streptavidin-C1-conjugated magnetic beads. Mix the solution thoroughly by placing the tube containing magnetic beads on the rotator at normal speed and room temperature for 30 minutes. Next, place the beads on the magnetic rack for three minutes at room temperature or until they gather on one side of the tube.
Gently remove the supernatant and add one milliliter of wash buffer one. Rotate the tube for two minutes on a rotator and repeat the step performed on the magnetic rack to remove the supernatant. Similarly, perform the washes with one milliliter of wash buffers two and three as previously demonstrated.
Next, remove the detergent in the wash buffers by adding 1.7 milliliters of 50 millimolar tris hydrochloride and repeating the step performed on the magnetic rack. Give six washes of two minutes each using one milliliter of 50 millimolar ammonium bicarbonate buffer. Once done, add 50 microliters of the protein extract containing 50 millimolar tris hydrochloride, 12%sucrose, 2%lithium lauryl sulfate, and 1.5%dithiothreitol to the beads, and place the tube in the incubator at 100 degrees Celsius for five minutes to give heat shock.
After repeating the step on a magnetic rack, store the supernatant at minus 80 degrees Celsius for LCMS/MS analysis. The western blot analysis of the extracted proteins showed successful protein expression and biotinylation in the infiltrated cucumber leaves. The higher expression level of labeled proteins and extra bands compared to the control confirmed the successful tagging of the target proteins of the gene of interest by AirID.
The results were confirmed using Anti-Flag and Anti-Mass antibodies showing the target band with an Anti-Flag antibody in all the transformed samples. After enriching biotinylated proteins with Streptavidin-C1-conjugated magnetic beads, multiple proteins of different sizes were observed. From all the results, it was concluded that AirID is a novel and ideal enzyme for analyzing protein-protein interactions in plants.
During proximity-labeling experiment, it is important to remember that five micromolar biotin and HR duration are ideal. The protocol outline the step by step setup of AirID based proximity labeling in plant, including preparation of the leaf sample, removing pre-biotin, quantifying extract protein, and enriching biotinylated proteins. AirID is projected to improve PPI independent biotinylation accuracy in combination.
It is concluded that AirID is ideal for PPI analysis in plants.
