2.4 Transgenic
Arabidopsis Plants for
Protein
Interaction Tests
4–5-week-old plants (ideally grown in 9-h day chamber at 22
C)
with 10–12 plants for each transgenic line and controls.
1. Test lines: Homozygous Arabidopsis transgenic plants expressing bait of interest tagged with BirA* under the control of a
DEX-inducible promoter (see Note 5).
2. Negative controls:
(a) Homozygous Arabidopsis transgenic plants expressing
BirA* alone under the control of a dexamethasoneinducible promoter (see Notes 6 and 7).
(b) Arabidopsis WT plants, e.g., Col-0 (see Note 8).
2.5 Biotin Infiltration
of Arabidopsis Plants
1. 20 mM Biotin (20 mL stock): 100 mg D-Biotin, 2 mL of 30%
NH 4 OH, 18 mL of 1 N HCl. After dissolving biotin in
NH 4 OH, add 5 mL of 1 N HCl at a time, wait 5 min on ice,
and repeat for a total of 18 mL. This solution can be stored for
months at 4
C. Use 2 mM (in H 2 O) working solution for
experiments.
2. 1 mL Syringe.
3. Paper tissues.
4. Goggles.
2.6 Dexamethasone
Induction of pDB
Constructs
1. 20 mM DEX stock solution in ethanol (can be stored at
À20
C for months)—use 30 μM working solution in H 2 O
for experiments.
2. Sharp forceps.
3. 50 mL Conical tubes.
4. Paper towels.
5. Liquid nitrogen and À80
C freezer.
2.7 Protein
Extraction
1. Protein extraction buffer (no detergents): 20 mM Tris–HCl
pH 8.0, 100 mM NaCl, Mass Spec-grade H 2 O, 1 mM DTT,
1% plant protease inhibitor cocktail.
2. Protein extraction buffer (with detergents): 20 mM Tris–HCl
pH 8.0, 100 mM NaCl, Mass Spec-grade H 2 O, 1 mM DTT,
1% plant protease inhibitor cocktail, sodium dodecyl sulfate
(SDS) to a concentration of 0.4% from a 10% stock solution,
Triton-X100 to a concentration of 1% in the protein extraction
buffer.
3. Equipment for extractions:
(a) Mortar and pestle.
(b) Liquid nitrogen.
(c) 50 mL conical tubes.
(d) Spatulas.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
431
Arabidopsis Plants for
Protein
Interaction Tests
4–5-week-old plants (ideally grown in 9-h day chamber at 22
C)
with 10–12 plants for each transgenic line and controls.
1. Test lines: Homozygous Arabidopsis transgenic plants expressing bait of interest tagged with BirA* under the control of a
DEX-inducible promoter (see Note 5).
2. Negative controls:
(a) Homozygous Arabidopsis transgenic plants expressing
BirA* alone under the control of a dexamethasoneinducible promoter (see Notes 6 and 7).
(b) Arabidopsis WT plants, e.g., Col-0 (see Note 8).
2.5 Biotin Infiltration
of Arabidopsis Plants
1. 20 mM Biotin (20 mL stock): 100 mg D-Biotin, 2 mL of 30%
NH 4 OH, 18 mL of 1 N HCl. After dissolving biotin in
NH 4 OH, add 5 mL of 1 N HCl at a time, wait 5 min on ice,
and repeat for a total of 18 mL. This solution can be stored for
months at 4
C. Use 2 mM (in H 2 O) working solution for
experiments.
2. 1 mL Syringe.
3. Paper tissues.
4. Goggles.
2.6 Dexamethasone
Induction of pDB
Constructs
1. 20 mM DEX stock solution in ethanol (can be stored at
À20
C for months)—use 30 μM working solution in H 2 O
for experiments.
2. Sharp forceps.
3. 50 mL Conical tubes.
4. Paper towels.
5. Liquid nitrogen and À80
C freezer.
2.7 Protein
Extraction
1. Protein extraction buffer (no detergents): 20 mM Tris–HCl
pH 8.0, 100 mM NaCl, Mass Spec-grade H 2 O, 1 mM DTT,
1% plant protease inhibitor cocktail.
2. Protein extraction buffer (with detergents): 20 mM Tris–HCl
pH 8.0, 100 mM NaCl, Mass Spec-grade H 2 O, 1 mM DTT,
1% plant protease inhibitor cocktail, sodium dodecyl sulfate
(SDS) to a concentration of 0.4% from a 10% stock solution,
Triton-X100 to a concentration of 1% in the protein extraction
buffer.
3. Equipment for extractions:
(a) Mortar and pestle.
(b) Liquid nitrogen.
(c) 50 mL conical tubes.
(d) Spatulas.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
431