Li Lab Protocols

Proximity Labeling (wheat seedling)

Day 1

  1. Sow the control seeds and transgenic seeds in a small box and grow the seedlings for 7-10 days in the greenhouse.
  2. Cut the seedlings from the basal and put the plant into a new 50 mL centrifuge tube with 50 μM biotin solution, making sure all the tissues can be submerged in the biotin buffer during the labeling.
  3. Vacuum 3 times, each time 10 min.
  4. Remove the biotin solutions and use ddH2O to wash the tissues 5 times.
  5. Use a paper towel to make the tissues dry, and immediately throw the tissue into the liquid nitrogen, and store in a -80℃ freezer.

Day 2

  1. Use liquid nitrogen (make sure tissues do not get thawed) to grind the plant tissue finely and collect 2g of ground powder into a new 50 mL tube.
  2. Add 4 mL of Extraction Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1 % SDS, 1 % Triton-X-100 or 1% NP-40, 0.5 % Na-deoxycholate, 1 mM EGTA, 1 mM DTT, 1x complete protease, 50 µM MG-132, 1 mM PMSF) to each tube.
  3. Stir the tissues with a clean spatula. Vortex in a cold room.
  4. Incubate on a rotor wheel at 4℃ for 10 min.
  5. Sonicate the tissue suspension on ice (Branson, 20% amplitude, 10 sec on / 10 sec off for 2 min)
  6. Centrifuge down the suspension at 2,500 rpm for 5 min at 4℃. Transfer the supernatant to a new tube.
  7. Centrifuge down the supernatant once again at 12,000g for 10 min at 4℃.
  8. Transfer ~ 5 mL of supernatant to new tubes for each sample and save 50 µL for western blotting. [Optional: Bio-Rad Protein Assay]
    1. Dilute Dye Concentrate to â…•X with water (Dark brick red color to light brown color)
    2. Transfer 250 L of diluted dye to different tubes.
    3. Add 5 µL of BSA – the color changes from light brown to blue
    4. Test supernatants (5 µL to new 250 µL of diluted dye solution)
  9. (Column Prep) Equilibrate the PD10 column with 5 mL of cold Equilibration Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1 % SDS, 1 % Triton-X-100 or 1% NP-40, 0.5 % Na-deoxycholate, 1 mM EGTA, 1 mM DTT) 5 times.
  10. Load 2.5 mL of the protein extracts on the PD-10 desalting column to remove excess free biotin.
  11. Elute the protein extract with 3.5 mL of equilibration buffer and collect the elite.
  12. Wash the column with 50 mL of Wash Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 % Triton-X-100 or 1% NP-40, 1 mM EGTA, 1 mM DTT).
  13. Repeat steps 9 - 11 with the remainder of the protein extracts (2.5 mL)
  14. Combine the fractions (3.5 mL + 3.5 mL).15. Take 50 µL out for western and Bio-Rad assay.
  15. (Bead Prep) Wash 200 µL of bead slurry (Dynabeads M280 or MyOne Streptavidin C1 (Invitrogen)) with extraction buffer 3 times.
  16. Transfer the beads to the protein extract (from step 14).
  17. Add 70 µL of 50 X protease inhibitor (to make the final concentration of 1X)
  18. Add 35 µL of 100 mM PMSF (to make the final concentration of 1 mM)
  19. Incubate the protein extract with beads on the rotor wheel at 4℃ overnight.

Day 3

  1. Spin down the beads at 1500 rpm at 4℃ for 2 mins. Keep the flow through and freeze to -80℃
  2. Wash the beads with 1 mL of each of the following solutions by incubating on the rotor wheel for 8 mins and removing the wash solution on a magnetic rack:
    1. 2x with cold extraction buffer (Transfer the beads into a new tube the first time
    2. 2x with cold equilibration buffer.
    3. 1x with cold 1 M KCl (and transfer the beads into a new tube)
    4. 2x with cold extraction buffer.
  3. Save 2% of the beads for quality control immunoblots. For the QC, boil the beads in 75 μL of 3X protein loading buffer supplemented with 20 mM DTT and 2 mM biotin, run on SDS-PAGE gel, and stain with Coomassie staining.
  4. Resuspend in 1 mL fresh extraction buffer and transfer to a new tube.
  5. Ship to Mass Spec Facility on ice (so that the samples are chilled but not frozen) and prepare for LC-MS/MS analysis.
Rapid DNA extraction from wheat leaf tissue
  1. Put a 2 cm-length wheat leaf into a 2 mL centrifuge tube and add 350 μL of TPS Extraction Buffer (1M KCl, 10 mM EDTA pH 8.0, 100 mM Tris-HCl pH 8.0) buffer and two steel beads.
  2. Use a tissue grinder at the high-frequency condition to grind the leaf for 2-3 cycles, for each cycle 30 sec.
  3. Place the grounded leaf into a 65°C oven for 30 min to fully denature the proteins.
  4. Centrifuge the leaf mixture at 12,000 rpm for 10 min at room temperature.
  5. Transfer the supernatant to a new 1.5 mL centrifuge tube, add two volumes of cold alcohol, mix well, and incubate it in a -20°C freezer for at least 30 min.
  6. Centrifuge at 12,000 rpm for 10 min, slowly removing the supernatant.
  7. Add 500 μL of 75% ethanol washing solution, thoroughly shake using a vortex mixer, then centrifuge at 12,000 rpm for 2 min and discard the supernatant.
  8. Repeat step 7, then air dry the centrifuge tube.
  9. Add 150 μL of ddH2O or TE buffer, dissolve it in a 65°C oven for 15 min, and store in a -20°C freezer.
Wheat protein extraction
  1. Collect the wheat leaves and grind them into a fine powder with liquid nitrogen. Place the powdered leaves into 1.5 mL centrifuge tubes and store them in a -80℃ freezer.
  2. Add 1.5 times protein Extraction Buffer (50 mM Tris-MES pH8.0, 10 mM EDTA pH8.0, 0.5 M Sucrose, 1 mM MgCl2,1 mM DTT, and 1 mM PMSF) to each sample. Immediately vortex to resuspend all the powder, then incubate on ice for about 30 minutes.
  3. Centrifuge the protein mixture at 4°C, 12,000 rpm for 10 minutes. Transfer the supernatant into a new 1.5 mL tube and add the 5×SDS loading buffer into each tube, boiling the sample by the dry heater at 95℃ for more than 5 min at least, and storing the samples in a -20°C freezer.
Wheat protoplast preparaction and transformation
  1. Sow the wheat seeds in a small box, let all the seeds germinate, and grow in a greenhouse for 7-10 days.
  2. Remove the roots and the top leaves, retaining only the tender leaf and coleoptile. Apply a small amount of 0.4 M mannitol solution on the seedlings to be cut, use a sharp knife to cut the seedlings into 0.1 mm pieces, and place the chopped tissue into a 250 mL triangular flask, containing 25 mL of Protoplast Enzyme Solution (20 mM MES pH 5.7, 0.4 M Mannitol, 20 mM KCl, 1.5% Cellulose R-10, 0.75% Macerozyme R-10, 10 mM CaCl2, 0.1% BSA)
  3. Vacuum the chopped tissue for 30 min. Incubate in the dark at room temperature on a horizontal shaker at 50 rpm for 3-5 hours for enzymatic digestion.
  4. Add 25 mL of W5 Buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES pH 5.7) to stop the enzymatic digestion, and continue to shake for 10 min.
  5. Gently transfer the supernatant into a new 50 mL round-bottom centrifuge tube by filtering a 40 µM filter.
  6. Centrifuge 100 g for 5 min at 4℃ (set acceleration and brake at level 1). Remove the supernatant and add 20 mL of W5 buffer to resuspend the protoplast.
  7. Add 20 mL of W5 buffer to the flask and shake the flask at 50 rpm for 10 min. Centrifuge 100 g for 5 min at 4℃ (set acceleration and brake as 1 level). Remove the supernatant
  8. Repeat step 7 twice.
  9. Remove the supernatant and resuspend the protoplasts in a proper volume of MMG Buffer (0.4 M Mannitol, 100 mM MgCl2, 4 mM MES pH 5.7).
  10. Add 10-20 μg of plasmid to a 2 mL centrifuge tube. Add 1000 μL of the resuspended protoplasts to the tube, then quickly add 1000 μL of PEG/Ca2+ Buffer (0.4 M Mannitol, 100 mM CaCl2, 25% PEG 4000). Immediately and gently invert the tube to mix, and incubate in the dark at room temperature for 25 min.
  11. Add 2000 μL of W5 buffer to the tube and mix by gentle inverting. Centrifuge at 100 g for 5 min (set the acceleration and brake to level 1), discard the supernatant, and resuspend the protoplasts in 1000 μL of W5 solution to remove any residual PEG/Ca2+ buffer.
  12. Incubate in the dark at 25°C for 16-18 h.