Plant Protein Purification
- Preparations before starting purification:
- Cool the centrifuge at 4ºC
- Prepare Extraction buffer (EB), Wash Buffer (WB) and Cleavage buffer (CB)
- Wash IgG beads with EB without protease inhibitors (three-four times), resuspend them in EB with protease inhibitors and aliquot in the 96-well plates; keep them at 4ºC until ready to use.
Use 5ml slurry for 2x 96-well plates.
- The purification should be done in the cold room; bring everything necessary to cold room before starting extraction: filter plates, pipettes, tips, buffers, balance, lids for plates, etc.
- Transfer the frozen tissue from -80ºC to ice and add EB. To ~ 0.8 ml frozen tissue add 1.5 ml EB with PI.
1.6 X 100 = 160 ml EB/96-well plate.
Leave on ice for 10-15 min. to thaw. Add zirconia beads to each tube. Place plates in the paint-shaker; shake 3 X 1 min. each with 1 min. on ice intervals.
- Spin for 10 min. at 40,000Xg to pellet cell debris in the cold centrifuge. Collect supernatant in the 96-well Whatman GF/C filter over prepared 96-well plates containing IgG beads (use 2 filters over two 96-well plates; try to pipette the extract in equal aliquots between the 2 filters, ~ 800 ul /well). Spin plates for 15 at 3,000Xg.
- Incubate the extract and IgG on a roller drum for 2 hours at 4ºC. Use the roller with a 360º rotation for the best mixing.
- After 2 hours, spin plates for 5 min. in the cold centrifuge and remove supernatant. Wash beads once with 0.5 ml WB I with PI and twice with WB I without PI.
- Wash beads once with 1 ml CB and remove SN.
- Add 50 µl of CB containing the cleavage protease (100ul /10 ml CB / 2 plates). Incubate on the roller drum for 14-16 hours (can go O/N).
- The next day, spin briefly and transfer supernatant to Millipore filter plate and collect SN without beads in fresh robot-compatible (ABI) 96-well plates.
Plate nr.1: Add 100% glycerol to a final concentration of 30%. Aliquot into 3-4 PCR plates (50µl aliquots) and store immediately at -80ºC.
Plate nr.2: Filter through PALL filter plates (15-20 min at 3,000Xg, or until solution concentrates 2-3 times). Add 20 ul PBS containing 30% glycerol to surface of filters, pipet up and down several times and transfer protein concentrate to fresh ABI robot-compatible 96-well plate. Aliquot and freeze at -80ºC.
Extraction Buffer
| Final conc. | | Stocks | 100 ml | 200 ml | 150 ml |
| 100 mM | Tris-HCl pH 7.5 | 1 M | 10 ml | 20 ml | 15 ml |
| 100 mM | NaCl | 5 M | 2 ml | 4 ml | 3 ml |
| 5 mM | EDTA | 0.5 M | 1 ml | 2 ml | 1.5 ml |
| 5 mM | EGTA | 0.25 M | 2 ml | 4 ml | 3 ml |
| 10 mM | NaF | 1 M | 1 ml | 2 ml | 1.5 ml |
| 0.10% | Triton -X | 10% | 1 ml | 2 ml | 1.5 ml |
| 0.10% | B-ME | 100% | 100 µl | 200 µl | 150 µl |
| 10% | Glycerol | 100% | 10 ml | 20 ml | 15 ml |
| 1 mM | PMSF | 0.2 M | 500 µl | 1 ml | 750 µl |
| 0.1mM | Na3VO4 | 1 M | 10 µl | 20 µl | 30 µl |
| 10 mM | B-GPh | 1 M | 1 ml | 2 ml | 3 ml |
| 1X | Prot. Inhib. | | 1 tb. | 2.tb | ½ tb. |
| ddH2O | | 70 ml | 140 ml | 105 ml |
Washing Buffer I
| Final conc. | | Stocks | 100 ml | 400 ml |
| 100 mM | Tris-HCl pH 7.5 | 1 M | 10 ml | 400 ml |
| 100 mM | NaCl | 5 M | 10 ml | 4 ml |
| 5 mM | EDTA | 0.5 M | 1 ml | 4 ml |
| 5 mM | EGTA | 0.25 M | 2 ml | 8 ml |
| 10 mM | NaF | 1 M | 1 ml | 4 ml |
| 0.10% | Triton -X | 10% | 1 ml | 4 ml |
| 0.10% | B-ME | 100% | 100 µl | 400 µl |
| 10% | Glycerol | 100% | 10 ml | 40 ml |
| 1 mM | PMSF | 0.2 M | 500 µl | 2 ml |
| 0.1mM | Na3VO4 | 1 M | 10 µl | - |
| 10 mM | B-GPh | 1 M | 1 ml | - |
| 1X | Prot. Inhib. | | | |
| ddH2O | | 62 ml | 268 ml |
Cleavage Buffer
| Final conc. | | Stocks | 10 ml | 100 ml | 150 ml |
| 50 mM | Tris-HCl pH 7.0 | 1 M | 500 µl | 5 ml | 7.5 ml |
| 150 mM | NaCl | 5 M | 300 µl | 3 ml | 4.5 ml |
| 1 mM | EDTA | 0.5 M | 10 µl | 100 µl | 150 µl |
| 1 mM | DTT | 0.1 M | 100 µl | - | - |
| 0.10% | Triton -X | 10% | 100 µl | 1 ml | 1.5 ml |
| ddH2O | | 9.1 ml | 91 ml | 137 ml |
|
