15. Infiltrate up to 50 leaves for each construct to get ~5 g of
tissue. Also infiltrate the negative controls BirA* and WT
Arabidopsis.
16. Use separate conical tubes for each construct and apply similar
DEX treatment for all constructs including negative controls.
17. Samples can be stored for several months at À80
C.
18. Use equal amounts of tissue for all constructs.
19. Continuously cool mortar and pestle with liquid nitrogen in
between grinding to keep samples frozen.
20. Vortex thoroughly until the powder is completely thawed and
resuspended in the buffer.
21. May need multiple 2 mL tubes for the supernatants. A portion
of the supernatant can be set aside as total protein extract for
western blots.
22. For soluble baits, continue to Subheading 3.7.
23. You can freeze the samples at À80
C at this step and carry on
with the next steps the following day. Soluble and insoluble
fractions can also be kept separate for the following steps.
24. Removing excess biotin allows efficient pulldown. Excess free
biotin in the protein extract interferes with pulldown of biotinylated proteins by streptavidin beads.
25. Make sure the supernatant gets concentrated to ~500 μL. It
may take longer than 15 min.
26. We incubated the beads with the protein extract overnight, but
timing can be shortened to 2–3 h.
27. For western blots, boil streptavidin Sepharose beads in SDS
loading buffer with 50 μM biotin to release the proteins bound
to the beads.
28. For western blots, membranes should not be washed in milk or
other blocking solutions containing biotin. We used 5% bovine
serum albumin.
29. For western blots, use streptavidin-horseradish peroxidase
conjugate to detect biotinylated proteins.
30. Supernatant can be saved to run on western blot to confirm no
biotinylated proteins are left in the unbound fraction.
31. From here on, only use non-autoclaved microcentrifuge tubes
and filter tips to prevent contamination in mass spectrometry
from plastic polymers. Also, use mass spectrometry-grade H 2 O
for all solutions.
32. Aliquot out 1/10 of the total volume of beads to keep for
western blot as affinity purified fraction.
33. Drying down the peptides with no heat should take about 2 h.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
439
tissue. Also infiltrate the negative controls BirA* and WT
Arabidopsis.
16. Use separate conical tubes for each construct and apply similar
DEX treatment for all constructs including negative controls.
17. Samples can be stored for several months at À80
C.
18. Use equal amounts of tissue for all constructs.
19. Continuously cool mortar and pestle with liquid nitrogen in
between grinding to keep samples frozen.
20. Vortex thoroughly until the powder is completely thawed and
resuspended in the buffer.
21. May need multiple 2 mL tubes for the supernatants. A portion
of the supernatant can be set aside as total protein extract for
western blots.
22. For soluble baits, continue to Subheading 3.7.
23. You can freeze the samples at À80
C at this step and carry on
with the next steps the following day. Soluble and insoluble
fractions can also be kept separate for the following steps.
24. Removing excess biotin allows efficient pulldown. Excess free
biotin in the protein extract interferes with pulldown of biotinylated proteins by streptavidin beads.
25. Make sure the supernatant gets concentrated to ~500 μL. It
may take longer than 15 min.
26. We incubated the beads with the protein extract overnight, but
timing can be shortened to 2–3 h.
27. For western blots, boil streptavidin Sepharose beads in SDS
loading buffer with 50 μM biotin to release the proteins bound
to the beads.
28. For western blots, membranes should not be washed in milk or
other blocking solutions containing biotin. We used 5% bovine
serum albumin.
29. For western blots, use streptavidin-horseradish peroxidase
conjugate to detect biotinylated proteins.
30. Supernatant can be saved to run on western blot to confirm no
biotinylated proteins are left in the unbound fraction.
31. From here on, only use non-autoclaved microcentrifuge tubes
and filter tips to prevent contamination in mass spectrometry
from plastic polymers. Also, use mass spectrometry-grade H 2 O
for all solutions.
32. Aliquot out 1/10 of the total volume of beads to keep for
western blot as affinity purified fraction.
33. Drying down the peptides with no heat should take about 2 h.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
439