Measuring Microtubule Persistence Length Using a Microtubule Gliding Assay - Methods in Cell Biology - page 18

The following buffer stock solutions should be prepared and stored (storage tem-
perature is suggested) prior to beginning an experiment. Flash freeze all protein stock
solutions in liquid nitrogen prior to storing at 80 C.

Tubulin: 50 ml, 0.5 mg, in 80 mM PIPES with 0.2 mM GTP ( 80 C)

Glycerol-MT
buffer:

80 mM PIPES, 10 mM MgCl 2 , 1 mM EGTA, pH 6.7, 30% Glycerol
(4 C)

80 C) GTP: 5 ml, 150 mM in water, pH 7 (

80 C) Fluorescent GTP: 5 ml, 1 mM in water (

80 C) Paclitaxel: 10 ml, 4 mM in DMSO (

Assay buffer (AB): 50 mM imidiazole, 50 mM KCl, 4 mM MgCl 2 , 2 mM EGTA, pH 6.7

(4 C)

AB with 30% w/v sucrose (4 C)

AB 30%

sucrose:

þ

117 mg/ml (4 C) BSA:

80 C long term, 4 C short term) Biotin-LC-BSA: 100 ml, 2 mg/ml in AB (

80 C long term, 4 C short term) a-Casein: 250 ml, 5 mg/ml in AB (

20 ml, 10 mg/ml in AB (80 C long term, 4 C short term)

SA:

100 ml, 200 mM in water (20 C, use within a day of thawing)

DTT:

10 ml, ( Yildiz et al., 2003 )(80 C long term, 4 C short term) Oxygen

scavenger:

100 ml, 120 mg/ml in water (20 C long term) Glucose:

10 ml, 150 mM in water (80 C) ATP:

Kinesin: 5 ml, 1 mMinAB( 80 C, use within a day of thawing)

The oxygen scavenging solution is prepared as described in Yildiz et al. using
glucose oxidase and catalase.

2.2.1 Polymerization of fluorescently labeled microtubules
The purpose of this protocol is to polymerize microtubules with a fluorescent GTP
analog bound permanently between the b -tubulin of one dimer and a -tubulin of
the next dimer. The target fluorophore density is approximately one fluorophore
per micrometer of microtubule length. The steps of this part of the procedure follow:

80 C freezer, thaw rapidly in hand
(37 C) until just thawed, and then place on ice for 5 min.
2. Spin at 14k RPM for 20 min to remove insolubles.
3. Remove supernatant, add to 50 m l glycerol-MT buffer to make 100 m l, and mix
gently with pipette.
4. Divide 100 m l into two 50 m l aliquots in 1.5-ml microcentrifuge tubes.
5. Dilute GTP to 10 mM in MilliQ water or AB (e.g., 14 m l buffer to 1 m l GTP).
6. To one tube, add 0.5 m l fluorescent GTP and 1.0 m l 10 mM GTP to make 1:20
dye-GTP MTs.

1. Remove an aliquot of tubulin from the