3.6 Protein
Extractions
1. Transfer 50 mL conical tubes with leaves to liquid nitrogen
before protein extraction.
2. Set centrifuge at 4
C.
3. Measure out ~5 g of frozen tissue for extraction (see Note 18).
4. Prechill mortar and pestle using liquid nitrogen.
5. Grind 5 g of frozen tissue into fine powder using mortar and
pestle (see Note 19).
6. Transfer powder to a fresh prechilled 50 mL conical tube using
a prechilled spatula, then transfer conical tube back into liquid
nitrogen until all samples have been ground.
7. Take samples out of liquid nitrogen and keep on ice. Add 2 mL
of precooled protein extraction buffer (no detergents) (see
Subheading 2.7, step 1) and mix by vortexing thoroughly (see
Note 20). Keep on ice.
8. Centrifuge the conical tube at 3000 Â g for 15 min at 4
C.
9. Transfer supernatant to a 15 mL conical tube.
10. Centrifuge at 5000 Â g for 15 min at 4
C.
11. Transfer supernatant to 2 mL microcentrifuge tubes (see Note
21).
12. Centrifuge at 20,000 Â g for 30 min at 4
C. Save supernatant
from this fraction as the soluble fraction in a fresh 2 mL microcentrifuge tube (see Note 22).
13. Resuspend pellet from step 12 in 200 μL of protein extraction
buffer (with detergents) (see Subheading 2.7, step 2).
14. Vortex intermittently for 15 min; keep on ice.
15. Centrifuge at 20,000 Â g for 30 min at 4
C.
16. Transfer supernatant (insoluble fraction) to a fresh 2 mL
microcentrifuge tube and combine with the soluble fraction
from step 12 (see Note 23).
3.7 Removing Excess
Biotin from Protein
Extract
1. Transfer the combined supernatants from step 12 (soluble
fraction) and step 16 (insoluble fraction) to an Amicon
Ultra-4 column to wash out excess biotin (see Note 24).
2. Spin column at 3000 Â g for 15 min at 4
C (see Note 25).
3. Repeat washes four times with 4 mL of protein extraction
buffer (no detergents) by spinning at 3000 Â g at 4
C for
30 min each with the supernatant reduced to 1 mL after the
final wash.
4. Transfer the 1 mL supernatant with concentrated protein
remaining in the column to a fresh 2 mL microcentrifuge
tube and keep on ice.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
435
Extractions
1. Transfer 50 mL conical tubes with leaves to liquid nitrogen
before protein extraction.
2. Set centrifuge at 4
C.
3. Measure out ~5 g of frozen tissue for extraction (see Note 18).
4. Prechill mortar and pestle using liquid nitrogen.
5. Grind 5 g of frozen tissue into fine powder using mortar and
pestle (see Note 19).
6. Transfer powder to a fresh prechilled 50 mL conical tube using
a prechilled spatula, then transfer conical tube back into liquid
nitrogen until all samples have been ground.
7. Take samples out of liquid nitrogen and keep on ice. Add 2 mL
of precooled protein extraction buffer (no detergents) (see
Subheading 2.7, step 1) and mix by vortexing thoroughly (see
Note 20). Keep on ice.
8. Centrifuge the conical tube at 3000 Â g for 15 min at 4
C.
9. Transfer supernatant to a 15 mL conical tube.
10. Centrifuge at 5000 Â g for 15 min at 4
C.
11. Transfer supernatant to 2 mL microcentrifuge tubes (see Note
21).
12. Centrifuge at 20,000 Â g for 30 min at 4
C. Save supernatant
from this fraction as the soluble fraction in a fresh 2 mL microcentrifuge tube (see Note 22).
13. Resuspend pellet from step 12 in 200 μL of protein extraction
buffer (with detergents) (see Subheading 2.7, step 2).
14. Vortex intermittently for 15 min; keep on ice.
15. Centrifuge at 20,000 Â g for 30 min at 4
C.
16. Transfer supernatant (insoluble fraction) to a fresh 2 mL
microcentrifuge tube and combine with the soluble fraction
from step 12 (see Note 23).
3.7 Removing Excess
Biotin from Protein
Extract
1. Transfer the combined supernatants from step 12 (soluble
fraction) and step 16 (insoluble fraction) to an Amicon
Ultra-4 column to wash out excess biotin (see Note 24).
2. Spin column at 3000 Â g for 15 min at 4
C (see Note 25).
3. Repeat washes four times with 4 mL of protein extraction
buffer (no detergents) by spinning at 3000 Â g at 4
C for
30 min each with the supernatant reduced to 1 mL after the
final wash.
4. Transfer the 1 mL supernatant with concentrated protein
remaining in the column to a fresh 2 mL microcentrifuge
tube and keep on ice.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
435