Spinach Rubisco

Procedures

Day 1

Prepare buffers and solutions filtered with a 0.22-uM filter and cooled to 4 degrees (except (NH4)2SO4).

Resuspension/lysis: 50 mM Tris-Cl (pH 7.6) and 0.2 mM EDTA

Anionic exchange A: 25 mM Tris-Cl (pH 7.6) and 0.1 mM EDTA

Anionic exchange B: 25 mM Tris-Cl (pH 7.6), 0.1 mM EDTA, and 1 M KCl

Size exclusion: 25 mM Tris-Cl (pH 7.6), 0.1 mM EDTA, and 150 mM KCl

1 L Millipore water

1 L 20% ethanol

200 mL 4.1 M (NH4)2SO4 (pH 7.0) (NH4OH)

5 mL 100 mM PMSF in isopropyl alcohol (stored at -20 degrees)

Day 2

  1. Mix the following in a 150-mL beaker and stir at 4 degrees: 120 mL of lysis buffer, 1 Roche protease inhibitor tablet, 1.2 mL of 100 mL PMSF, and 2.4 g (2% w/v) PVPP.

  2. Add solution to pre-chilled blender and mix it well. Slowly add powdered frozen leaf tissue. Grid tissue with 15-sec bursts, then additional 15-sec bursts.

  3. Filter homogenates through 2 layers of miracloth, and recover as much as possible by gently squeezing. Miracloth should be washed with Milli-Q water and stored at 4 degrees beforehand.

  4. Centrifuge the sample at 17,000 rpm for 30 minutes at 4 degrees. Keep the supernatant.

  5. Ammonium sulfate precipitation: add 83.45 mL saturated NH4OH dropwise to sample supernatant on ice with constant stirring over 30-minute period. (NH4)2SO4 volume to add = 0.45 / volume of supernatant

  6. Centrifuge it at 17,000 rpm for 30 minutes at 4 degrees.

  7. Add 51.76 mL saturated NH4OH dropwise to sample supernatant over 15-minute period.

  8. Centrifuge at 17,000 rpm for 30 minutes at 4 degrees and save pellet.

  9. Resuspend the pellet to a maximum volume of 10 mL anionic exchange buffer A.

  10. Load it onto 4 pre-equilibrated PD-10 columns.

  11. Elute with 3.5 mL of anionic exchange buffer A for each PD-10 columns.

  12. Equilibrate HiTrap Q HP column with the anionic exchange buffer A.

  13. Use a syringe to load diluted sample onto a HiTrap Q HP column.

  14. Run AKTA protocol -

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