S10-Z + X-S11 + GFP1–9 + p19 (more stringent negative
control 2)
Z-S11 + GFP1–10 + p19 (Z-S11 tagging test)
S10-Z + S11-GFP1–9 + p19 (S10-Z tagging test)
Only in certain special cases, the N-terminally fused S11
tag and the C-terminally fused S10 tag can also be used for
detecting PPIs depending on the geometry of interaction;
however, such an orientation is not necessarily taken as a routine for PPI analysis.
3. We routinely check the positive Agrobacterium transformant
that harbors desired constructs by using both colony PCR and
restriction analysis. After extracting plasmid DNA from Agrobacterium culture and retransforming it to E. coli, we pick two
E. coli colonies grown on LB plate containing appropriate
antibiotics. Only after confirming that both E. coli colonies
are correct, we will maintain the positive Agrobacterium clone
in glycerol stock.
4. To prepare a final 4-mL infiltration mixture enough for infiltration of 3–4 leaves, we recommend to mix Agrobacterium cultures in 2 Â 2-mL Eppendorf tube, each at OD 600 suspension of
0.25 in equal proportion to reach an OD 600 final at 0.0625 for
each strain. For example, in one set of co-expression combination in a 2-mL Eppendorf tube, mix 0.5 mL of p19, 0.5 mL of
S10-X, 0.5 mL of Y-S11, and 0.5 mL of GFP1–9 for PPI
analysis; mix 0.5 mL of p19, 0.5 mL of Y-S11, 0.5 mL of
GFP1–10, and 0.5 mL of infiltration medium for Y-S11 tagging test. An OD 600 final of each strain lower than 0.0625 can
also be tested if a lower level of protein expression is desired.
5. The standard microscopic analysis can be performed 1 day after
induction, corresponding to 2 days post-infiltration. However,
for new constructs, signals can be monitored every 24 h up to
3 days post-induction, corresponding to 4 days postinfiltration. If a lower level of protein expression is desired, a
weaker induction can also be tested, either by the dilution of
β-Estradiol concentrations or shortening the induction time.
Acknowledgments
This work was supported by grants from the Ministry of Science
and Technology of the Republic of China (MOST 103-2311-B007-012-MY2 and MOST 105-2621-M-007-001-MY3). I also
appreciate Ms. Chang-Yi Chiu’s help with the proof-reading and
for some comments on the manuscript.
334
Tzu-Yin Liu
control 2)
Z-S11 + GFP1–10 + p19 (Z-S11 tagging test)
S10-Z + S11-GFP1–9 + p19 (S10-Z tagging test)
Only in certain special cases, the N-terminally fused S11
tag and the C-terminally fused S10 tag can also be used for
detecting PPIs depending on the geometry of interaction;
however, such an orientation is not necessarily taken as a routine for PPI analysis.
3. We routinely check the positive Agrobacterium transformant
that harbors desired constructs by using both colony PCR and
restriction analysis. After extracting plasmid DNA from Agrobacterium culture and retransforming it to E. coli, we pick two
E. coli colonies grown on LB plate containing appropriate
antibiotics. Only after confirming that both E. coli colonies
are correct, we will maintain the positive Agrobacterium clone
in glycerol stock.
4. To prepare a final 4-mL infiltration mixture enough for infiltration of 3–4 leaves, we recommend to mix Agrobacterium cultures in 2 Â 2-mL Eppendorf tube, each at OD 600 suspension of
0.25 in equal proportion to reach an OD 600 final at 0.0625 for
each strain. For example, in one set of co-expression combination in a 2-mL Eppendorf tube, mix 0.5 mL of p19, 0.5 mL of
S10-X, 0.5 mL of Y-S11, and 0.5 mL of GFP1–9 for PPI
analysis; mix 0.5 mL of p19, 0.5 mL of Y-S11, 0.5 mL of
GFP1–10, and 0.5 mL of infiltration medium for Y-S11 tagging test. An OD 600 final of each strain lower than 0.0625 can
also be tested if a lower level of protein expression is desired.
5. The standard microscopic analysis can be performed 1 day after
induction, corresponding to 2 days post-infiltration. However,
for new constructs, signals can be monitored every 24 h up to
3 days post-induction, corresponding to 4 days postinfiltration. If a lower level of protein expression is desired, a
weaker induction can also be tested, either by the dilution of
β-Estradiol concentrations or shortening the induction time.
Acknowledgments
This work was supported by grants from the Ministry of Science
and Technology of the Republic of China (MOST 103-2311-B007-012-MY2 and MOST 105-2621-M-007-001-MY3). I also
appreciate Ms. Chang-Yi Chiu’s help with the proof-reading and
for some comments on the manuscript.
334
Tzu-Yin Liu