UBQ10:sXVE:ER-GFP1–10; #125664
UBQ10:sXVE:S11-GFP1–9; #125665
UBQ10:sXVE:ER- S11-GFP1–9; #125666
2. It is recommended to evaluate the topology, subcellular localization, and targeting signals of membrane proteins of interest.
Wrong tag orientation may affect protein localization or reduce
the expression level. For analyzing PPI of protein X and
protein Y, construct combinations for a single standard tripartite split-sfGFP assay which contains fusion protein tagging
tests and possible negative controls are listed below. More
stringent negative controls can also be included if appropriate
information is available. For example, point mutations or deletion variants that disrupt the interaction interface of fusion
proteins can be used. Alternatively, using a Z protein which is
known to localize to the same subcellular compartment of PPI
is also suitable. By either way, the expression level of negative
controls can be examined in tagging tests.
Orientation A:
S10-X + Y-S11 + GFP1–9 + p19
S10-X + ATG-3 Â HA-S11 + GFP1–9 + p19 (standard negative control 1)
S10–3 Â HA + Y-S11 + GFP1–9 + p19 (standard negative
control 2)
S10-X + S11-GFP1–9 + p19 (S10-X tagging test)
Y-S11 + GFP1–10 + p19 (Y-S11 tagging test)
S10-X + Z-S11 + GFP1–9 + p19 (more stringent negative
control 1)
S10-Z + Y-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)
Orientation B:
S10-Y + X-S11 + GFP1–9 + p19
S10-Y + ATG-3 Â HA-S11 + GFP1–9 + p19 (standard negative
control 1)
S10–3 Â HA + X-S11 + GFP1–9 + p19 (standard negative
control 2)
S10-Y + S11-GFP1–9 + p19 (S10-Y tagging test)
X-S11 + GFP1–10 + p19 (X-S11 tagging test)
S10-Y + Z-S11 + GFP1–9 + p19 (more stringent negative
control 1)
Tripartite Split-sfGFP Assay
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