Biology Reference
In-Depth Information
protein purification is consistently not working, you should try changing
buffers and baculovirus. We have found removing the unbound protein in
batch results in better protein than column purification. Perhaps, this is due to
the length of time it takes to run a column.
13.1.2 Required elements for biophysical assays
13.1.2.1 Microtubules
Microtubules are the substrate of microtubule-severing enzymes. We prepare several
different types of microtubules in order to determine if these AAA
enzymes rec-
ognize differences in the nucleotide state of the polymer, shifts in the number of pro-
tofilaments, or have a preference for the plus- or minus-end. To do this, we make
microtubules stabilized with Taxol or guanalyl-( a , b )-methylene diphosphate
(GMPCPP). We also use polarity-marked microtubules and microtubules with pro-
tofilament defects that are visualizable via fluorescence.
We purify and prepare our own tubulin from pig brains following the protocol
from Shelanski ( Shelanski, Gaskin, & Cantor, 1973 ) and Hyman ( Hyman et al.,
1991 ). We use TRITC rhodamine-labeled tubulin (Cytoskeleton, Catalog number
TL590M, Denver, CO) or in-house-labeled tubulin. In-house labeling is done as de-
scribed in Hyman et al. (1991) . We use DyLight 650, DyLight 550, or DyLight 488
with NHS ester (Pierce/Thermo Scientific, 46402, Rockford, IL).
þ
13.1.2.1.1 Buffers, reagents, and equipment
13.1.2.1.1.1 Buffers
￿ PEM-100: 100 mM pipes, 2 mM EGTA, 2 mM MgCl 2 , pH 6.9 with KOH.
13.1.2.1.1.2 Reagents
￿ Unlabeled tubulin (5 mg/ml)
￿ Labeled tubulin (5 mg/ml)
￿ Paclitaxel (Taxol) (Sigma-Aldrich, T7402, St. Louis, MO)
￿ Guanosine 5 0 -triphosphate sodium salt hydrate (GTP) (Sigma-Aldrich, G8877,
St. Louis, MO)
￿ GMPCPP (Sigma-Aldrich, M3170, St. Louis, MO)
13.1.2.1.1.3 Equipment
￿ Micro ultracentrifuge
￿ Micro ultracentrifuge tubes
￿ Tabletop temperature-controlled centrifuge
13.1.2.1.2 Detailed procedures
13.1.2.1.2.1 Taxol-stabilized microtubules
1. Mix unlabeled tubulin (5 mg/ml) with labeled tubulin at any percentage from 5%
to 20% labeling. If using lyophilized rhodamine tubulin from cytoskeleton, we
recommend hydrating in PEM-100 and incubating for 10 min on ice before
combining in order to dissolve the protein fully.
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