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 -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 robotcompatible 96-well plate. Aliquot and freeze at -80ºC.
Extraction buffer:
Final
conc.
100 mM
100 mM
5 mM
5 mM
10 mM
0.10%
0.10%
10%
1 mM
0.1mM
10 mM
1X
Tris-HCl pH
7.5
NaCl
EDTA
EGTA
NaF
Triton -X
B-ME
Glycerol
PMSF
Na3VO4
B-GPh
Prot. Inhib.
ddH2O
Stocks
100 ml
200 ml
150 ml
1M
5M
0.5 M
0.25 M
1M
10%
100%
100%
0.2 M
1M
1M
10 ml
2 ml
1 ml
2 ml
1 ml
1 ml
100 µl
10 ml
500 µl
10 µl
1 ml
1 tb.
70 ml
20 ml
4 ml
2 ml
4 ml
2 ml
2 ml
200 µl
20 ml
1 ml
20 µl
2 ml
2.tb
140 ml
15 ml
3 ml
1.5 ml
3 ml
1.5 ml
1.5 ml
150 µl
15 ml
750 µl
30 µl
3 ml
½ tb.
105 ml
Stocks
100 ml
400 ml
1M
5M
0.5 M
0.25 M
1M
10%
100%
100%
0.2 M
1M
1M
10 ml
10 ml
1 ml
2 ml
1 ml
1 ml
100 µl
10 ml
500 µl
10 µl
1 ml
400 ml
4 ml
4 ml
8 ml
4 ml
4 ml
400 µl
40 ml
2 ml
-
Washing buffer I:
Final
conc.
100 mM
100 mM
5 mM
5 mM
10 mM
0.10%
0.10%
10%
1 mM
0.1mM
10 mM
1X
Tris-HCl pH
7.5
NaCl
EDTA
EGTA
NaF
Triton -X
B-ME
Glycerol
PMSF
Na3VO4
B-GPh
Prot. Inhib.
ddH2O
62 ml
268 ml
Cleavage buffer:
Final conc.
50 mM
150 mM
1 mM
Tris-HCl
pH 7.0
NaCl
EDTA
Stocks
10 ml
100 ml
150 ml
1M
5M
0.5 M
500 µl
300 µl
10 µl
5 ml
3 ml
100 µl
7.5 ml
4.5 ml
150 µl
1 mM
0.10%
DTT
Triton -X
ddH2O
0.1 M
10%
100 µl
100 µl
9.1 ml
1 ml
91 ml
1.5 ml
137 ml