8. Gently resuspend washed protoplasts in 10–20 mL Buffer A
and aliquot 1 mL each into 14 mL round-bottom polystyrene
or glass test tubes (see Note 5). One tube will be used for each
transformation. Centrifuge protoplasts at 80 Â g, 20
C, 5 min.
Remove 850 μL of the supernatant and gently swirl the protoplasts in the remaining supernatant. The protoplasts are now
ready to be transformed with plasmid DNA and should be kept
at RT and used immediately.
9. The DNA transformation reactions must be carried out in
about a minute for each transformation event, without interruptions (see Note 6). Take a test tube with protoplasts in one
hand and start to gently shake the tube in a circular motion.
With the other hand, add 10 μL of plasmid DNA (encoding a
fusion of the carotenoid enzyme with a fluorescent-tagged
protein, or with one half of YFP) while gently shaking in
circular motions. In the case of BiFC, immediately add 10 μL
of the second plasmid DNA (encoding a fusion of a carotenoid
enzyme with the second half of YFP), while still shaking gently.
While shaking the tube, slowly add 500 μL Buffer C, measured
using a cut pipette tip to accurately dispense this viscous solution, and continue shaking for 10 s. Stop shaking and put the
tube in a rack. Immediately and gently add 4.5 mL of Buffer D,
cap the tube and do not shake or move the tube. Repeat for all
the other transformation reactions. Leave the tubes in a rack for
20–30 min undisturbed.
10. After the incubation is over, add another 5 mL of Buffer A and
gently invert the tubes upside down several times. Centrifuge
at 80 Â g, 20
C, 5 min. Discard the supernatant, and wash
with 5 mL of Buffer A. Discard the supernatant and gently
resuspend each protoplast sample in 1 mL of the Buffer A using
cut tips to gently resuspend the protoplasts. Transfer the transformed protoplasts into the wells of a 24-well plate if an
inverted microscope is used (see Note 7), and incubate overnight at 21–25
C in the dark or dim light (see Note 8).
11. Samples are then visualized by fluorescent microscopy and
compared with controls to determine where the proteins are
localized or if they interact. For these experiments, a confocal
fluorescent microscope, such as the inverted DM16000B with
TCS SP8 system and LAS AF software (Leica Microsystems
CMS, Germany) is recommended. Confocal laser microscopes
provide visualization at Z-planes (e.g., several “layers” of the
image that can be fused together by the software into a 3D
image for better visualization), which is especially important
for analysis and detailed localization of the enzymes in plastids.
An oil or water immersion objective 63–100Â is required for
optimal resolution. In confocal microscopy, a 488 nm argon
laser is used to excite fluorescence of GFP and chlorophyll; the
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Maria Shumskaya et al.
and aliquot 1 mL each into 14 mL round-bottom polystyrene
or glass test tubes (see Note 5). One tube will be used for each
transformation. Centrifuge protoplasts at 80 Â g, 20
C, 5 min.
Remove 850 μL of the supernatant and gently swirl the protoplasts in the remaining supernatant. The protoplasts are now
ready to be transformed with plasmid DNA and should be kept
at RT and used immediately.
9. The DNA transformation reactions must be carried out in
about a minute for each transformation event, without interruptions (see Note 6). Take a test tube with protoplasts in one
hand and start to gently shake the tube in a circular motion.
With the other hand, add 10 μL of plasmid DNA (encoding a
fusion of the carotenoid enzyme with a fluorescent-tagged
protein, or with one half of YFP) while gently shaking in
circular motions. In the case of BiFC, immediately add 10 μL
of the second plasmid DNA (encoding a fusion of a carotenoid
enzyme with the second half of YFP), while still shaking gently.
While shaking the tube, slowly add 500 μL Buffer C, measured
using a cut pipette tip to accurately dispense this viscous solution, and continue shaking for 10 s. Stop shaking and put the
tube in a rack. Immediately and gently add 4.5 mL of Buffer D,
cap the tube and do not shake or move the tube. Repeat for all
the other transformation reactions. Leave the tubes in a rack for
20–30 min undisturbed.
10. After the incubation is over, add another 5 mL of Buffer A and
gently invert the tubes upside down several times. Centrifuge
at 80 Â g, 20
C, 5 min. Discard the supernatant, and wash
with 5 mL of Buffer A. Discard the supernatant and gently
resuspend each protoplast sample in 1 mL of the Buffer A using
cut tips to gently resuspend the protoplasts. Transfer the transformed protoplasts into the wells of a 24-well plate if an
inverted microscope is used (see Note 7), and incubate overnight at 21–25
C in the dark or dim light (see Note 8).
11. Samples are then visualized by fluorescent microscopy and
compared with controls to determine where the proteins are
localized or if they interact. For these experiments, a confocal
fluorescent microscope, such as the inverted DM16000B with
TCS SP8 system and LAS AF software (Leica Microsystems
CMS, Germany) is recommended. Confocal laser microscopes
provide visualization at Z-planes (e.g., several “layers” of the
image that can be fused together by the software into a 3D
image for better visualization), which is especially important
for analysis and detailed localization of the enzymes in plastids.
An oil or water immersion objective 63–100Â is required for
optimal resolution. In confocal microscopy, a 488 nm argon
laser is used to excite fluorescence of GFP and chlorophyll; the
230
Maria Shumskaya et al.