10. The expression can be induced 1 day post-infiltration by painting β-Estradiol in EtOH on the abaxial side of the leaves at a
concentration range of 2.5–10 μg/mL (9.175–36.7 μM;
1:1000–1:4000 dilution of stock solution). Depending on
the level of protein expression, additional days of β-Estradiol
treatment can be applied once daily on consecutive days.
3.4 Microscopic
Imaging and Tripartite
Split-GFP Assay
1. Excise ~1-cm
2 segments (without veins) from infiltrated leaves.
For image comparison, only use sample from the same leaf area
of leaves of the same age. Immediately place a drop of water on
a microscope slide and transfer a leaf sample with the abaxial
side up on water drop. Cover the sample with a cover-glass and
ensure that there are no air-bubbles between sample and coverglass.
2. Mount the microscope slide onto a confocal microscope with
the abaxial side of the sample toward the objective.
3. For comparison of tripartite split-sfGFP association efficiencies
of different construct combinations, image all samples using
identical settings for GFP channel and the frequency of cells
displaying complemented split-sfGFP can be calculated by
counting the number of fluorescing cells per square centimeters. This is particularly simple for PPI analysis at the endoplasmic reticulum, which is in irregular shapes in epidermal
cells. Alternatively, measure mean fluorescence intensities of
entire subcellular organelles, such as nuclei, chloroplast, and
mitochondria, in randomly taken images from different areas of
each sample using the microscope software or Fiji. The value of
fluorescence intensities obtained for GFP1–10 or S11-GFP1–9
tagging test could be defined as 100% and thus used as an
adequate readout for normalizing tripartite split-sfGFP
association.
4 Notes
1. The β-Estradiol inducible tripartite split-sfGFP-based binary
vectors with Addgene number are listed below.
UBQ10:sXVE:S10-(MCS); #108177
UBQ10:sXVE:S10-(MCS)-3 Â HA; #108178
UBQ10:sXVE:(MCS)-S11; #108179
UBQ10:sXVE:(MCS)-3 Â HA-S11; #108180
UBQ10:sXVE:ATG-3 Â HA-S11; #108238
UBQ10:sXVE:GFP1–9; #108187
UBQ10:sXVE:GFP1–10; #125663
332
Tzu-Yin Liu
concentration range of 2.5–10 μg/mL (9.175–36.7 μM;
1:1000–1:4000 dilution of stock solution). Depending on
the level of protein expression, additional days of β-Estradiol
treatment can be applied once daily on consecutive days.
3.4 Microscopic
Imaging and Tripartite
Split-GFP Assay
1. Excise ~1-cm
2 segments (without veins) from infiltrated leaves.
For image comparison, only use sample from the same leaf area
of leaves of the same age. Immediately place a drop of water on
a microscope slide and transfer a leaf sample with the abaxial
side up on water drop. Cover the sample with a cover-glass and
ensure that there are no air-bubbles between sample and coverglass.
2. Mount the microscope slide onto a confocal microscope with
the abaxial side of the sample toward the objective.
3. For comparison of tripartite split-sfGFP association efficiencies
of different construct combinations, image all samples using
identical settings for GFP channel and the frequency of cells
displaying complemented split-sfGFP can be calculated by
counting the number of fluorescing cells per square centimeters. This is particularly simple for PPI analysis at the endoplasmic reticulum, which is in irregular shapes in epidermal
cells. Alternatively, measure mean fluorescence intensities of
entire subcellular organelles, such as nuclei, chloroplast, and
mitochondria, in randomly taken images from different areas of
each sample using the microscope software or Fiji. The value of
fluorescence intensities obtained for GFP1–10 or S11-GFP1–9
tagging test could be defined as 100% and thus used as an
adequate readout for normalizing tripartite split-sfGFP
association.
4 Notes
1. The β-Estradiol inducible tripartite split-sfGFP-based binary
vectors with Addgene number are listed below.
UBQ10:sXVE:S10-(MCS); #108177
UBQ10:sXVE:S10-(MCS)-3 Â HA; #108178
UBQ10:sXVE:(MCS)-S11; #108179
UBQ10:sXVE:(MCS)-3 Â HA-S11; #108180
UBQ10:sXVE:ATG-3 Â HA-S11; #108238
UBQ10:sXVE:GFP1–9; #108187
UBQ10:sXVE:GFP1–10; #125663
332
Tzu-Yin Liu