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Box 2. Preparation of celery juice extract • TIMING ∼10 h

From the following article

A protocol for TILLING and Ecotilling in plants and animals

Bradley J Till, Troy Zerr, Luca Comai and Steven Henikoff

Nature Protocols 1, 2465 - 2477 (2006)

doi:10.1038/nprot.2006.329

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  1. Wash approx1 lb (one bunch) of celery in cool water. Remove any leafy material to aid in juicing. Pass the celery through a juicer. Approximately 400 ml of juice should be produced.
    filled triangle CRITICAL Perform all steps at 4 °C.
  2. Centrifuge the juice for 20 min at 2,600g to pellet debris. Transfer the supernatant to a new tube.
  3. Add 1 M Tris and PMSF to the cleared celery juice so that the final concentration of the solution is 0.1 M Tris-HCl, pH 7.7, 100 muM PMSF.
    ! CAUTION PMSF is hazardous. Wear gloves; avoid direct contact with skin or inhalation.
  4. Bring the supernatant to 25% (NH4)2SO4 by adding 144 g l-1 of solution. Mix gently at 4 °C for 30 min. Centrifuge the supernatant at 13,000–16,200g at 4 °C for 40 min. Discard the pellet.
  5. Bring the supernatant from 25 to 80% (NH4)2SO4 by adding 390 g l-1 of solution. Mix gently at 4 °C for 30 min. Spin at 13,000–16,200g for 1.5 h. Save the pellet and discard the supernatant, being careful in decanting the supernatant.
    filled square PAUSE POINT The pellet from Step 5 can be stored at -80 °C for at least 2 weeks.
  6. Suspend the pellets in approx 1/10 the starting volume (e.g., 40 ml) with Tris/KCl/PMSF buffer, ensuring the pellet is completely dissolved.
  7. Dialyze thoroughly against Tris/KCl/PMSF buffer at 4 °C for 1 h. Use dialysis membrane with a 10,000 kDa cutoff size. Use at least 4 liter of buffer per 20 ml of enzyme suspension. It may be easiest to split the suspension into several dialysis tubes. After 1 h, replace buffer with fresh Tris/KCl/PMSF buffer. Repeat two more times for a total of four buffer changes.
    filled square PAUSE POINT After two buffer changes, dialysis can be left overnight. Dialysis can proceed as long as 1 week without any apparent loss in enzyme activity. Four hours of dialysis can be taken to be the minimal amount of dialysis required.
  8. If desired, clear the dialyzed solution by spinning at 10,000g for 30 min. Greater than 90% of the enzyme activity should be retained in the soluble fraction.
    filled square PAUSE POINT Aliquot into small volumes (approx1 ml) and store at -80 °C. This protein mixture does not require storage in glycerol and remains stable through multiple freeze–thaw cycles. When activity is determined, working stock volumes can be aliquoted (e.g., enough for 1 week) and stored at -20 °C. One pound of celery should produce enough enzyme for approximately 500,000 reactions.