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7. Texas Red hydrazide (TxRed, 90% single isomer) (Life Technologies, CA)
8. 7-Hydrazinyl-4-methylcoumarin (coumarin hydrazine, CH)
Note : This fluorophore is not commercially available, but it is not complicated
to synthesize ( Banerjee, Panosian, et al., 2010 ). A commercially available
hydrazide such as 7-(diethylamino)coumarin-3-carbohydrazide, CZ, can be
substituted for CH.
9. 4-Amino- DL -phenylalanine hydrate; 97% (4aF) (Acros Organic, NJ)
1.1.3 Detection of labeled tubulin
1. Sample buffer, 3
stock (3.75 mL glycerol, 3.0 mL 10% (w/v) SDS (Catalog #
L5750, Sigma-Aldrich), 0.3 mL 0.5% (w/v) bromophenol blue, 0.325 mL
water, 1.875 mL, 0.5 M Tris, pH 6.8). Add 7.5%
b
-mercaptoethanol to the
sample buffer prior to running the gel
2. SDS-PAGE gel (8% acrylamide resolving layer and 6% acrylamide stacking
layer)
3. Resolving buffer (1.5 M Tris, pH 9.8)
4. Stacking buffer (0.5 M Tris, pH 6.8)
5. PBST (10 mM phosphate buffer at pH 7 with 0.1% Tween-20)
6. Blocking buffer (PBST with 5% bovine serum albumin)
7. Monoclonal anti-tubulin, tyrosine antibody TUB-1A2 (Sigma-Aldrich)
8. Streptavidin-HRP (Life Technologies)
1.1.4 Detection of polymerization activity
1. Taxol
2. DMSO (Sigma)
1.2 METHOD
1.2.1 Protein isolation and purification
1.2.1.1 Tubulin
Isolate bovine brain tubulin by two cycles of assembly/disassembly followed by
phosphocellulose chromatography ( Williams & Lee, 1982 ). Add 1-2 mM MgSO 4
and drop-freeze the purified tubulin into liquid nitrogen and store until use. Gently
thaw the frozen pellets and equilibrate the protein in PME buffer. We use Sephadex
G-50 in 1 mL syringe spin columns following the method of Penefsky (1979) .
Determine tubulin concentration spectrophotometrically by using an extinction
coefficient of 1.23 (mg/mL) 1 cm 1 at 278 nm in PME.
Owing to the conserved nature of both tubulin and the ligase, tubulin from other
sources can be substituted for bovine brain tubulin. We have successfully performed
labeling procedure on chicken erythrocyte tubulin, purified as described previously
( Sharma, et al., 2010 ).
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