This mystery can help answer a few questions in the field of cell biology such as protein input into chloroplasts. The main advantage of this technology is that protein input into chloroplasts can be studied under en vivo conditions. Demonstrating the procedure will be Junho Lee and Hyangju Kang, two post outs from my laboratory.
To begin, harvest intact leaf tissues from two-week-old plants from one to two B5 plates. Use a surgical knife to harvest the leaves and place them into a 50 milliliter conical tube containing 25 milliliters of enzyme solution. Place the tube horizontally on a rotary shaker and incubate with gentle agitation at 22 degrees Celsius in the dark for eight to 12 hours until only petioles and stems remain in the solution.
After completing incubation, filter the enzyme solution containing released protoplasts through a 140 micrometer pore size mesh into a fresh Petri dish. Then carefully layer the solution on top of 15 milliliters of 21%sucrose solution in a 50 milliliter conical tube. Centrifuge the tube in a swinging bucket rotor at 98 gs for 10 minutes with the lowest acceleration and deceleration settings.
After that, use patura pipette to carefully removed the protoplasts from the uppermost layer containing enzyme solution and from the interface between the enzymes solution and sucrose solution. Transfer this solution to a 50 milliliter conical tube containing 30 milliliters of W5 solution and invert the tube to mix. Centrifuge the tube at 51 gs for six minutes.
Use a pipette to discard this supernatant carefully without disturbing the protoplasts in the pellet. Add 25 milliliters of W5 solution and gently re-suspend the protoplasts. For stabilization, incubate in a four Celsius refrigerator for a minimum of one hour.
After four degree Celsius incubation, pellet the protoplasts completely by centrifuging them at 46 gs for two minutes. Carefully remove the supernatent completely and add MANG solution to the protoplast pellet to achieve five times ten to the six per milliliter. Use a pipette to add 10 micrograms of the plasma DNA and 300 microliters of protoplast solution to a fresh 13 milliliter round bottomed tube.
Mix by gently rotating the tubes by hand and then immediately add 300 microliters of freshly prepared 40%PEG solution. Mix completely by tilting the tube almost horizontally and rotating it gentle several times by hand. Incubate at room temperature for 30 minutes.
Then add one milliliter of W5 solution and mix completely by gently rotating the tube by hand as previously. Incubate the sample for 10 minutes at room temperature. Repeat two more times with adding first 1.5 milliliter and then two milliliters of W5 and after the final addition and mixing, incubate for 30 minutes.
Centrifuge the tube at 46 gs for four minutes and then discard the supernatant. Add two milliliters of W5 solution to the pellet and mix gently, but completely, in the same manner as previously. Incubate at 22 degrees Celsius in a dark chamber for 18 hours.
To analyze the protein import using florescence microscopy, use a pipette with a trimmed tip to place 10 microliters of the protoplast solution on a glass slide. Use a cover slip to carefully cover the solution to avoid damaging the protoplasts. Place the slide on the stage of a fluorescence microscope with an excitation admission filter that is set for observing green fluorescent protein and chlorophyll autofluorescence.
Use a cooled charge couple device camera to capture images and process them using an imagine editing software to produce pseudo-color images. For total protein extraction and immunoblotting, remove one milliliter from the protoplast solution and mix the remaining one milliliter. Transfer this protoplast solution into a centrifuge tube and centrifuge at 46 gs for four minutes.
Remove the supernatant after completing centrifugation and add 80 microliters of denaturation buffer. Vortex vigoriously for five seconds. Add five XDS sample buffer, mix well and boil for 10 minutes to denature.
Proceed with standard STS page and immunoblotting with anti-GFP antibody. RbcS and TGFT, a fusion construct encoding the 79 end terminal amino acid residues of RBCS containing the transit peptide fused to GFP, was used to study the import of proteins into chloroplasts by florescence microscopy and immunoblotting. When examined by florescence microscopy, green fluorescent signals from the target protein merged with the red fluorescent signals from chlorophyll autoflorescence indicating protein import into chloroplasts.
After isolating total protein, two protein bands are observed in the immunoblot, showing that a protein was properly imported into chloroplasts. The upper band corresponds to the full length precursor and the lower band to the processed form after import into chloroplasts. The amount of the processed form of the protein increased in a time-dependent manner, suggesting that the protein is imported into chloroplasts.
At this development, this technique paved the way for researchers in the field of cell biology to explore protein tiring or membrane thread picking in Arabidopsis.
