Biology Reference
In-Depth Information
GMPCPP-microtubules are prepared as described earlier, with the modification that
the GMPCPP-microtubules should be prepared with a higher ratio of fluorescently
labeled tubulin, creating bright GMPCPP-MTs and dim paclitaxel-microtubules.
Combine both microtubules into a single tube and introduce the mixture to the flow
chamber. The mixing ratio that gives a desirable number of paclitaxel- and
GMPCPP-microtubules in each field of view should be determined experimentally
each day. At this point, fluorescent MAPs of interest can be introduced, and prefer-
ential binding to subsets of the mixed, paclitaxel-microtubules can be observed
(
Bechstedt & Brouhard, 2012
). If such preferential binding occurs, the bright and
dim subsets can be compared to the 14-pf GMPCPP-microtubules as a reference.
21.2.5
Purification of DCX-GFP for labeling 13-pf microtubules
DCX is a MAP that binds preferentially to 13-pf microtubules within certain concen-
tration ranges. The following purification describes how to obtain human DCX (Acc
Nr. NP_835365) containing an N-terminal 6xHis tag and C-terminal coding se-
quence for EGFP and StrepTagII (
Bechstedt & Brouhard, 2012
).
Inoculate 2 ml LB medium with a colony from a plate of freshly transformed
BL21(DE3) bacteria and incubate with agitation (250 rpm) for
14 h at 25
C.
Use this overnight culture to inoculate 1 L LB medium at 37
C and let cells grow
to OD 0.4-0.6. Cool cells down and induce at 18
C with 1 mM IPTG. Incubate over-
night (
14 h) with agitation (250 rpm) for protein expression. Spin down expression
cultures at 5000
g
for 15 min and reconstitute pellets in 4 ml/g buffer A. Pellets can
80
C when not directly processed. For DCX purification, lyse the pel-
lets using, for example, an Emulsiflex C3 (Avestin). Clear the lysate by centrifuga-
tion at 45,000
be stored at
g
for 1 h. Incubate the supernatant with mild agitation with 1 ml
His60 Ni resin for 30 min at 4
C. Wash the resin twice with buffer A and load
the resin on a disposable plastic column. Elute the bound protein, including DCX-
GFP, with four column volumes of buffer B. Load the green-fluorescent elution
fractions on a 1-ml Strep-tactin Superflow column. Wash the column twice with
two column volumes Strep-tactin wash buffer. Elute the purified DCX-GFP in
0.5 ml fractions with Strep-tactin elution buffer. Peak fractions can be combined
and either used fresh or flash-frozen in 20
m
l aliquots and stored at
80
C.
DCX-GFP should be introduced to a mixed population of paclitaxel-microtubules
with 14-pf GMPCPP-microtubules present as an internal control. Serial dilutions of
DCX-GFP should be tested, as DCX-GFP binds equivalently to all microtubule types
at very low concentrations (
<
1 nM) and saturates all microtubule types at very high
concentrations (
1
m
M). For example, prepare a solution of 0.1
m
M DCX-GFP in
imaging buffer: BRB80
>
antifade reagents;
this solution is introduced into the microscope chamber and images are recorded.
The serial dilutions will identify a concentration range at which a subset of the
paclitaxel-microtubules is brighter than the 14-pf GMPCPP-microtubules and the
dim subset of the paclitaxel-microtubules. This concentration of DCX-GFP can then
be used in future experiments to label the 13-pf fraction.
þ
10
m
M paclitaxel
þ
0.1 mg/ml BSA
þ
