2. Fill a 1 mL hypodermic syringe with the infiltration mixture.
Infiltrate leaves from the abaxial side. For each infiltration
mixture use three plants. Infiltrate three leaves per plant. Into
the left side of the leaf infiltrate the wild-type sensor-containing
mixture, while into the right side of the same leaf introduce the
mutant sensor-containing mixture (Fig. 1b). Choose leaves
that are fully expanded and avoid the wrinkled ones, which
are highly resistant to infiltration.
3. Grow the infiltrated plants at 25
C under long-day conditions
for 72 h before analyzing protein expression.
3.5 Analysis
of Protein Expression
As a measure of gene silencing, expression of the wild-type sensor/
mutant sensor-encoded GFP-Renilla luciferase fusion protein is
analyzed by quantitative Western blotting. Western blot analysis
of proteins is composed of three consecutive steps. First the proteins are separated according to their apparent molecular masses
using SDS-PAGE. Next, the gel is electro-transferred to nitrocellulose or PVDF membranes. Generally, nitrocellulose membranes
give lower background than PVDF membranes; however the latter
is more durable. Therefore, if multiple rounds of stripping/immunological detection are planned, PVDF membranes should be used
instead of nitrocellulose. The final step is the immunological detection of the protein to be studied. Since Western blot analysis is a
routine technique in most molecular biology laboratories, no
detailed protocols for SDS-PAGE and electro-transfer of gels are
provided here. Instead, the reader is referred to a number of excellent laboratory manuals [14, 15]. Only the protein lysate preparation and immunological detection/quantitation of signals are
described here in more details.
3.5.1 Preparation
of Protein Lysates from
Infiltrated Leaves
1. Punch 1 cm diameter disks from both sides of infiltrated leaves.
Pool disks from the same side of leaves infiltrated with the same
suspensions (total of nine disks).
2. Collect the disks into a porcelain mortar. Freeze disks by pouring a small volume of liquid N 2 into the mortar and quickly
grind them into a fine powder using a porcelain pestle.
3. Add 1800 μL of ice-cold lysis buffer to the mortar and continue homogenization for 30 s.
4. Pour lysate into a 2 mL tube. Keep tubes on ice until all samples
are collected.
5. Centrifuge lysates at 20,000 Â g for 20 min at 4
C.
6. Transfer supernatants into clean tubes.
7. Store samples at À70
C until further analysis.
444
Ka ´ roly Fa ´ tyol
Infiltrate leaves from the abaxial side. For each infiltration
mixture use three plants. Infiltrate three leaves per plant. Into
the left side of the leaf infiltrate the wild-type sensor-containing
mixture, while into the right side of the same leaf introduce the
mutant sensor-containing mixture (Fig. 1b). Choose leaves
that are fully expanded and avoid the wrinkled ones, which
are highly resistant to infiltration.
3. Grow the infiltrated plants at 25
C under long-day conditions
for 72 h before analyzing protein expression.
3.5 Analysis
of Protein Expression
As a measure of gene silencing, expression of the wild-type sensor/
mutant sensor-encoded GFP-Renilla luciferase fusion protein is
analyzed by quantitative Western blotting. Western blot analysis
of proteins is composed of three consecutive steps. First the proteins are separated according to their apparent molecular masses
using SDS-PAGE. Next, the gel is electro-transferred to nitrocellulose or PVDF membranes. Generally, nitrocellulose membranes
give lower background than PVDF membranes; however the latter
is more durable. Therefore, if multiple rounds of stripping/immunological detection are planned, PVDF membranes should be used
instead of nitrocellulose. The final step is the immunological detection of the protein to be studied. Since Western blot analysis is a
routine technique in most molecular biology laboratories, no
detailed protocols for SDS-PAGE and electro-transfer of gels are
provided here. Instead, the reader is referred to a number of excellent laboratory manuals [14, 15]. Only the protein lysate preparation and immunological detection/quantitation of signals are
described here in more details.
3.5.1 Preparation
of Protein Lysates from
Infiltrated Leaves
1. Punch 1 cm diameter disks from both sides of infiltrated leaves.
Pool disks from the same side of leaves infiltrated with the same
suspensions (total of nine disks).
2. Collect the disks into a porcelain mortar. Freeze disks by pouring a small volume of liquid N 2 into the mortar and quickly
grind them into a fine powder using a porcelain pestle.
3. Add 1800 μL of ice-cold lysis buffer to the mortar and continue homogenization for 30 s.
4. Pour lysate into a 2 mL tube. Keep tubes on ice until all samples
are collected.
5. Centrifuge lysates at 20,000 Â g for 20 min at 4
C.
6. Transfer supernatants into clean tubes.
7. Store samples at À70
C until further analysis.
444
Ka ´ roly Fa ´ tyol