Biology Reference
In-Depth Information
To increase fluorescence signal, proteins are often tagged with multiple tandem cop-
ies of YFP. A straightforward method to accomplish this uses a selectable, integrat-
ing plasmid containing tandem copies of YFP. A C-terminal fragment of the targeted
gene, selected to contain
50 bp on either side of a restriction site unique to the plas-
mid, is cloned, without the stop codon, in-frame upstream of the YFPs. Plasmid di-
gestion at the unique restriction site generates a linear fragment that will be targeted
to the C-terminus of the endogenous locus. Amajor advantage of this approach is that
tandem incorporation of multiple plasmid copies will simply generate additional
C-terminal YFP regions, which lack the promoter and most of the ORF and, thus,
are unlikely to have residual expression/activity. For an example in which the kinesin
Kip3 was tagged by fusion with 3YFP (see
Gupta, Carvalho, Roof, & Pellman,
2006
).
The positive YFP-fusion transformants should be identified microscopically or
by PCR as described above. The forward primer should anneal to a region of the
tagged protein that was not cloned into the plasmid, and the reverse primer to the
linker sequence between the tagged protein and the YFPs. This avoids complications
from primer annealing to the repetitive sequences within the tandem YFPs. Generate
clonal strains and confirm the YFP fusion before storing and/or using for analyses.
>
22.6.2
Maintaining equal levels of YFP-fusion proteins
Strains used for quantitative analyses must be verified by western blot to express
equal amounts of the YFP-tagged protein. Prepare log-phase cultures in SC as
described above.
Note
: We find improved consistency in quantitative imaging and western blots by
preparing cultures under selective pressure for the tagged protein, for example,
SC-Leu for cells containing
KIP3-3YFP-LEU2
.
Determine OD600 of the cultures, centrifuge 10 ml of cells, and resuspend in 1 ml of
cold 0.1 M NaOH. Incubate 10 min on ice, centrifuge, and resuspend in
l
SDS-PAGE loading buffer. The exact volume of loading buffer should reflect the
OD600 ratio to maintain equal cell-to-volume ratios among the samples. Heat to
100
C for 10 min, cool on ice, and centrifuge 10 min at 13,000
200
m
g
. Use equal vol-
umes for western blotting. For low abundance fusion proteins, we find 4-12% Bis-
Tris gradient gels (NuPAGE, Invitrogen) useful to concentrate and sharpen protein
bands. GFP/CFP/YFP can be detected with polyclonal anti-GFP antibody (ABM).
Actin (anti-
b
-actin, Abcam) can be used to verify equal loading.
22.6.3
Microscopy and analysis of microtubule-associated proteins
Prepare concentrated log-phase cells, mount on agarose pads, and image in the CFP
and YFP channels, essentially as described for GFP-Tub1. The z-series should be
acquired rapidly to minimize microtubule movements during imaging. Acquisition
speeds can generally be improved by capturing the entire CFP z-series before switch-
ing filters to capture YFP, increased excitation intensity, a piezoelectric z-control,
2
2 binning, and increased efficiency with dedicated CFP/YFP filter sets rather
