Biology Reference
In-Depth Information
using seed aliquots, thaw them at room temperature and keep them warm. Do not at-
tempt to thaw seeds on ice or they will depolymerize. When inspecting seeds, the
dye will bleach fairly quickly under standard TIRF conditions (3 mW laser power,
500 ms exposure, 37
C). A variation in the above protocol that is fairly often used
is to add taxol as a seed stabilizing agent. However, we do not recommend this pro-
cedure as it may, later on, also interfere with the dynamic behavior of MTs synthesized
from the seeds.
8.1.4
TIRF microscopy of
TIPs in vitro
The following assay allows the study of the MT system and its associated binding
partners in a cell-free manner. One major advantage of this system is that the MT
interacting protein of interest is studied independently of complicating
in vivo
fac-
tors. In effect, it is possible to dissect the interplay between the binding partner and
dynamic MTs
in vitro
, that is, where does the partner bind, for how long, and how
does it affect the MT. With the protein of interest purified (and tagged with a fluor-
ophore), all that is required is to have the components for MT growth ready and add
them to the prepared sample chambers. TIRF microscopy is then used to image the
action of the proteins. With respect to protein tags and fluorophores, it is worth men-
tioning that a recent study (
Zhu et al., 2009
) found that the presence of the hexa-HIS
tag had an effect on the binding of EB1 to MTs (non-HIS-tagged protein bound with
a lower affinity compared to the standard HIS-tagged EB1). These studies were per-
formed using classic (or “bulk”) biochemistry approaches and not with the
in vitro
assay described here. Nevertheless, it is worth mentioning as a point of caution when
considering the purification of proteins that using the hexa-HIS tag may have an ef-
fect on protein binding to the MTs.
þ
Required materials
MRB80 buffer: 80 mM PIPES buffered to pH 6.8 with KOH (room
temperature), 1 mM EGTA, 4 mM MgCl
2
. On ice.
MRB80 buffer: 80 mM PIPES buffered to pH 6.8 with KOH (room
temperature), 1 mM EGTA, 4 mM MgCl
2
. Prewarmed to 37
C (require a
minimum of 500
l per
in vitro
sample).
Poly-
L
-lysine PEG biotin (PPL-PEG biotin): 0.2 mg ml
1
stock, dissolved in
MRB80 buffer. Surface solutions, PPL(20)-g[3.5]-PEG(2)/PEG(3.4)-biotin
(50%), 10 mg pack size. Aworking stock may be stored at 4
C, the remaining
solution can be stored at
m
80
C in 200
l aliquots.
Streptavidin (available from Sigma-Aldrich) or neutravidin (available from
Invitrogen). 1 mg ml
1
stock solution in MRB80 buffer. Stored at
m
80
Cas
20
m
l aliquots.
k
-Casein. Available from Sigma-Aldrich, purified from bovine milk. Catalog
number C0406. Stored as a 5 mg ml
1
stock solution in MRB80 buffer. 50
l
m
Aliquots.
