Plant protein purification.docVIP

Plant protein purification 1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. Wash beads once with 1 ml CB and remove SN. 7. 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). 8. 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 -80