3.2 Fixation
1. Harvest plants and immediately transfer to an Erlenmeyer
flask containing 200 mL of 1% formaldehyde. Place the Erlenmeyer flask into a desiccator and apply vacuum for 15 min (see
Note 3). Carefully release vacuum.
2. Terminate fixation by quenching: replace formaldehyde by
200 mL of 125 mM glycine. Apply vacuum for 5 min, carefully
release vacuum, and remove glycine solution.
3. Carefully wash plants with ice-cold sterile water four times.
4. Remove liquid by blotting onto absorbent paper tissue and
transfer plants to liquid nitrogen immediately. Store at
À80
C until further use.
3.3 Preparation
of Beads
1. To prepare Sepharose beads for preclearing, pipette 100 μL of
sepharose beads each (50% slurry) in an Eppendorf cap, add
1 mL ice-cold RIP-LB (without inhibitors), and incubate for
5 min at 4
C (see Note 4). Microcentrifuge for 1 min at 500 g
at 4
C. Repeat this washing step twice. Store at 4
C until use.
2. To prepare beads for immunoprecipitation, wash 20 μL of
GFP-Trap
® beads (50% slurry) with 1 mL ice-cold RIP-LB
buffer in an Eppendorf cap for 5 min at 4
C (see Note 5).
Microcentrifuge for 1 min at 500 g. Repeat this washing step
twice. Store at 4
C until use.
3.4 Total Extract
Preparation
1. Grind frozen plant material to a fine powder using a mortar and
pestle prechilled with liquid nitrogen (see Note 6).
2. Prepare 2 mL Eppendorf caps with punctured lid and prechill
in liquid nitrogen.
3. Transfer ground plant material corresponding to 0.5 g to prechilled 2 mL Eppendorf cap. Per immunoprecipitation use
2 Â 0.5 g plant powder distributed on two 2 mL
Eppendorf caps.
4. Remove Eppendorf caps with powder from liquid nitrogen and
add 750 μL of buffer RIP-LB prewarmed to 60
C to
each tube.
5. Add steel balls. Replace punctured lid by an intact lid of a new
tube. Shake tubes in an Eppendorf thermomixer at 40
C and
1400 rpm until a viscous homogenate is obtained.
6. Microcentrifuge at maximal speed in a microcentrifuge at 4
C
for 10 min.
7. Combine supernatants and filter the cell lysate extract using a
syringe and
8. 0.45 μm filter.
3.5 Immunoprecipitation
1. To preclear extracts for immunoprecipitation, transfer 1 mL of
the extract (see step 8 in Subheading 3.4) to 50 μL of washed
RIP-seq
457
1. Harvest plants and immediately transfer to an Erlenmeyer
flask containing 200 mL of 1% formaldehyde. Place the Erlenmeyer flask into a desiccator and apply vacuum for 15 min (see
Note 3). Carefully release vacuum.
2. Terminate fixation by quenching: replace formaldehyde by
200 mL of 125 mM glycine. Apply vacuum for 5 min, carefully
release vacuum, and remove glycine solution.
3. Carefully wash plants with ice-cold sterile water four times.
4. Remove liquid by blotting onto absorbent paper tissue and
transfer plants to liquid nitrogen immediately. Store at
À80
C until further use.
3.3 Preparation
of Beads
1. To prepare Sepharose beads for preclearing, pipette 100 μL of
sepharose beads each (50% slurry) in an Eppendorf cap, add
1 mL ice-cold RIP-LB (without inhibitors), and incubate for
5 min at 4
C (see Note 4). Microcentrifuge for 1 min at 500 g
at 4
C. Repeat this washing step twice. Store at 4
C until use.
2. To prepare beads for immunoprecipitation, wash 20 μL of
GFP-Trap
® beads (50% slurry) with 1 mL ice-cold RIP-LB
buffer in an Eppendorf cap for 5 min at 4
C (see Note 5).
Microcentrifuge for 1 min at 500 g. Repeat this washing step
twice. Store at 4
C until use.
3.4 Total Extract
Preparation
1. Grind frozen plant material to a fine powder using a mortar and
pestle prechilled with liquid nitrogen (see Note 6).
2. Prepare 2 mL Eppendorf caps with punctured lid and prechill
in liquid nitrogen.
3. Transfer ground plant material corresponding to 0.5 g to prechilled 2 mL Eppendorf cap. Per immunoprecipitation use
2 Â 0.5 g plant powder distributed on two 2 mL
Eppendorf caps.
4. Remove Eppendorf caps with powder from liquid nitrogen and
add 750 μL of buffer RIP-LB prewarmed to 60
C to
each tube.
5. Add steel balls. Replace punctured lid by an intact lid of a new
tube. Shake tubes in an Eppendorf thermomixer at 40
C and
1400 rpm until a viscous homogenate is obtained.
6. Microcentrifuge at maximal speed in a microcentrifuge at 4
C
for 10 min.
7. Combine supernatants and filter the cell lysate extract using a
syringe and
8. 0.45 μm filter.
3.5 Immunoprecipitation
1. To preclear extracts for immunoprecipitation, transfer 1 mL of
the extract (see step 8 in Subheading 3.4) to 50 μL of washed
RIP-seq
457