VARIABLES QUE AFECTAN LA LIXIVIACIÓN

ddH₂O at 1 ml/min. They were then equilibrated in modified column buffer (no NaCl,

pH 6.8 for the SP column, pH 7.9 for the Q column) by washing with 5 CV at 1 ml/min.

Further washings were performed by washing with elution buffer (5 CV modified column

buffer with 1 M NaCl, before re-equilibrating with 5 CV of modified column buffer. Protein

from the previous step was diluted 1:10 in modified column buffer and loaded by a sample

pump at 3 ml/min. The sample pump was washed with 5 ml of modified column buffer at

3 ml/min. The protein bound to the column was washed with 5 CV of modified column

buffer at 1 ml/min. The protein was eluted by salt gradient over 15 CV by linear change

from modified column buffer to elution buffer at 1 ml/min. Elutions were captured in 250µl

fractions When moving from the Q column to the SP column, the peak fractions (>50%

of the maximum) from absorbance at 488 nm was combined as the sample to be added

to the SP column. After use, the column was washed with a further 5 CV of elution buffer,

and re-equilibrated with 5 CV of modified column buffer at 1 ml/min. For storage, the

column was washed with 5 CV of ddH₂O or until the conductance was stable, and then

transferred into 20% ethanol.

If further concentration was required (<25µM for EB3, or <30µM for EB1 or EB2),

The peak fractions were combined and concentrated using vivaspin columns (Sartorius),

supplemented with 20% glycerol, snap frozen and stored in liquid nitrogen. Protein

concentration was determined by measuring absorption at 280nm and coomassie blue

staining of poly-acrylamide gel electrophoresis protein gels.

2.3

Purification of Tubulin from Porcine Brain

Tubulin was prepared from porcine brains according to published protocols (Gell et

al., 2011) with a couple of modifications. The brains were extracted immediately after

termination of the animal and placed into bags of ice cold PBS containing PBS ice cubes,

and transferred to the laboratory. The brains were normally within the laboratory within

two hours of termination. Upon arrival the brains were weighed and supplemented with

50% weight to volume crude buffer (100 mM PIPES pH 6.8, 0.5 mM MgCl₂, 2 mM EGTA

2.3. PURIFICATION OF TUBULIN FROM PORCINE BRAIN

anti-proteases) homogonised by blending at full power for 30 s (Kenwood Mixer). The

slurry was then clarified by centrifugation in SLA-1500 rotor (Thermo Scientific) at 14,500

rpm, 4◦C for 1 hour.

The tubulin was then subjected to its first polymerisation cycle. The supernatant was

transferred to a clean vessel and made up to 5 mM MgCl₂, 1 mM NaGTP and 50µl

DCI, 33% v/v glycerol. The solution was then raised quickly, under constant swirling,

to 37◦C and left incubating at 37◦C for an hour, with intermittent perturbation, to allow

microtubules to form. The microtubules were pelleted by centrifugation in SLA-1500 rotor

at 14,500 rpm, 37◦C for 3 hours. The supernatant was discarded and at 4◦C the pellets

were released from the centrifuge pot wall with 10 ml/pellet polymerisation buffer (100 mM

PIPES pH 6.8, 0.5 mM MgCl₂, 2 mM EGTA pH 8.0, 0.1 mM EDTA, 0.1 mM NaGTP, 4 mM

DTT supplemented with anti-proteases and 0.1%β-mercaptoethanol) and homogenised

by fifteen strokes of a tight Wheaton homogeniser on ice. The MT’s were then left to

depolymerise on ice for 40 minutes and the solution clarified by high speed centrifugation

in T-865 (Sorvall) at 65,000 rpm, 4◦C for 30 minutes.

The tubulin was then subjected to a second polymerisation cycle. The supernatant was

transferred to a clean vessel and made up to 5 mM MgCl₂, 1 mM NaGTP and 50µl DCI,

33% v/v glycerol. The solution was then raised, under constant swirling, to 37◦C and

left to incubate for an hour, with intermittent perturbation, to allow microtubules to form.

The microtubules were pelleted by centrifugation in T-865 rotor at 45,000 rpm, 37◦C for

1 hour. At this point the supernatant was discarded and the pellet snap frozen, and stored

at -80◦C.

In general at this point the frozen pellets were split in half and purified in two batches.

This was due to reduced capacity in the centrifugal rotas used below to process the

whole prep in one step.

The pellets were defrosted on ice and detached using 250µl of column buffer (50 mM

PIPES pH 6.9, 0.2 mM MgCl₂, 1 mM EGTA pH 8.0). Each pellet was transfer to a Wheaton

homogeniser with an additional 1 ml column buffer per pellet, and homogenised by ten

2.3. PURIFICATION OF TUBULIN FROM PORCINE BRAIN

depolymerise on ice for 40 minutes and the solution clarified by high speed centrifugation

in TLA-100.3 (Sorvall) at 50,000 rpm, 4◦C for 30 minutes. The clarified supernatant was

then loaded onto a phosphocellulose column equilibrated with GTP column buffer (see

below). The supernatant was loaded via sample loop at 0.2 ml/min with GTP column

buffer. The tubulin was eluted at 0.2 ml/min with GTP column buffer and collected in 1 ml

fractions. Following elution the peak fractions were combined, aliquoted and snap frozen

in liquid nitrogen.

If required the MAPs can be eluted/ or the column cleaned for a second round by running

2 CV of high salt column buffer at 1ml/min. The column was then equilibrated with

GTP column buffer, then ddH₂O, before being dismantled. The AKTA was washed and

returned to its storage state by rinsing with 20% EtOH.

2.3.1 Phosophocellulose Column Preparation

The phosphocellulose column was prepared by adding 20 g of phosphocellulose (P11,

Whatman) powder to 0.5 L of 0.5 M NaOH to create a uniform slurry. The resin was

allowed to settle for 5 minutes before the supernatant was removed. The solution was

then neutralised by the addition of 0.5 L of 0.5 M K-phosphate, pH 6.8. Neutralisation was

tested by pH paper. After allowing the resin to settle for 5 minutes the clear supernatant

was removed and the resin washed with 1 L of ddH₂O. The resin was then acid treated by

removing the clear supernatant after 5 minutes and re-suspending into 0.5 L 0.5 M HCl.

The resin was allowed to settle for 5 minutes before the clear supernatant was removed.

The solution was then neutralised by the addition of 0.5 L of 0.5 M K-phosphate, pH 6.8.

Neutralisation was tested by pH paper. After allowing the resin to settle for 5 minutes

the clear supernatant was removed and the resin washed with 0.5 L of ddH₂O, this step

was repeated once. The clear supernatant was removed after 5 minutes and the resin

was resuspended in column buffer. After 5 minutes half of the clear supernatant was

removed.

The resin was hand packed into a XK56 AKTA column by gravity flow using column buffer.

2.4. SODIUM DODECYL SULFATE - POLYACRYLAMIDE GEL