Download Kinesin Microtubule Gliding Assay 1. Make labeled microtubules

Kinesin Microtubule Gliding Assay
1. Make labeled microtubules:
 Mix 1.5 ul 50uM TMR-tubulin and 18.5 ul 17uM tubulin (1:5 molar ratio of labeled to
unlabeled). Ratios are reasonably approximate – 1.5ul of 2mg/ml TMR tubulin, 18.5ul of
3mg/ml unlabelled tubulin works fine.
 Add 20 ul 2X polymerization mix (see below) and incubate at 37 for 30 minutes
 Add 40 ul BRB80 (+1mM DTT and 20uM taxol). – allowing polymerisation without taxol
produces longer microtubules.
2. Make chambers
Use 18mm x 18mm x 1.5 coverslips. No need to acid wash them for these gliding assays. Lay two
strips of double-sided sticky tape on a slide with a gap of 3-4mm between them (~10 μl chamber).
Place the coverslip on top of the tape, press down firmly and then remove excess tape.
3. Make Brb80/Casein Buffer (BCB)
1 ml: 200ul 5X Brb80
1 ul DTT (1M stock in H2O)
2 ul taxol (10mM paclitaxel in DMSO)
50ul 25mg/ml Casein
747ul dH2O
4. Make Gloxy (oxygen scavenger – make fresh for very sensitive experiments, otherwise stored at 4oC
for up to a month)
50 ul 1x Brb80
15 ul Catalase (Stored at 4oC - Roche)
7.5 mg glucose oxidase (Stored at -20 - Sigma)
- Spin 5 minutes max speed microfuge OR Spin through 0.2 μm spin filter
5. Dilute GFP tagged kinesin motor into BCB
Typically 1ul motor:19ul of BCB – more motor if concentration is low or it binds poorly
6. Prepare Motility Buffer (Make fresh every hour):
 92ul BCB
 5ul
ATP (100mM Mg:ATP)
 1ul
glucose (1:100 of 450mg/ml)
 1ul
gloxy
 1ul
MTs
7. Chamber preparation:
 Add 1 chamber volume concentrated GFP ab (~400ug/ml) to chamber (about 10ul).
 Wash in 100ul BCB. Incubate 30-60sec.
 Add 20ul motor diluted in BCB (typically 1:20)
 Wash 2 X 100 ul BCB
 Flow in 20 ul motility buffer (see below)
8. Image on microscope: typically 100x objective, 1.5x optivar, 1-5second exposures
Modificiations to Motility Buffer
1) If you have a very slow moving motor it can be useful to flow in just MTs diluted in BCB and
allow them to bind (turn coverslip face down and wait a few minutes). Then flow in motility
mix containing everything except MTs. This will reduce the background fluorescence and
prevent extra MTs binding during data collection.
2) Velocity is very dependant on salt concentration. Increasing salt will increase velocity.
3) Different motors tend to prefer different buffers – simply swap out BRB80 for the appropriate
buffer.
4) If you want to use lower ATP concentrations or have a more controlled experimental conditions
try the following protocol (Phosphoenol pyruvate/Pyruvate kinase is an ATP regenerating system
which allows you to maintain a constant ATP concentration).
20ul
66ul
4ul
1ul
1ul
1ul
1ul
1ul
5ul
5x BRB80
dH2O
casein (25mg/ml)
glucose (1:100 of 450mg/ml)
gloxy
MTs
Pyruvate kinase (Roche)
200mM Phospho-enol-pyruvate
ATP (1mM Mg:ATP) - 50uM final – for accuracy pipet 5ul of different ATP dilutions.
Standard Buffers
BRB80 (1X):
80 mM PIPES,
1 mM MgCl2,
1 mM EGTA,
pH 6.8 with KOH
BRB80 (5X stock – store at 4oC)
60.4g PIPES,
2.5ml 1M MgCl,
12.5ml 0.2M EGTA
pH 6.8 with KOH
2xPolymerisation Mix:
1x BRB80
20% DMSO
2mM GTP
H2O
2ml 5x BRB80
2ml 100% DMSO
200 μl 100mM stock or 10.5mg solid
10ml
Casein for motility assays
Caesin: Sigma C-5890 (Stored with chemicals at room temperature)
1) Add 2g casein to 50ml of 20mM Tris pH 8.0
2) Check pH with indicator strips: probably about pH 7.0 – bring it up to pH 8.0 using NaOH
3) Leave on rotary wheel in cold room for ~1hour – recheck the pH (probably about 6.0) – and
readjust with NaOH to bring it back up to pH 8.0
4) Leave between 1 hour and overnight – recheck the pH again, adjust and leave another hour if
necessary.
5) Do a quick check of the protein concentration by Bradford (need it to be greater than 25mg/ml) –
Spin 50 ul of Casein solution in a microfuge, then take 1ul into 200ul Bradford, 800ul Water. If
the solution is bright blue (OD595 is greater than 1.0) then the concentration is high enough.
6) Transfer Casein to Ti70 tubes and spin 65,000 2 hours. Take off the clear middle layer (top layer
is a little bit cloudy)
7) Do a Bradford with a BSA standard to get the protein concentration, dilute to 25mg/ml with
20mM Tris pH 8.0.
8) Filter through a 0.2 micron filter. Aliquot and snap freeze in liquid nitrogen. Store at -80.
Notes:
Casein preparation is not as complicated as it looks from the above. Basically the key is to get
the Casein to dissolve to 30mg/ml. To do this you need to keep adjusting the pH back up to 8.0
using NaOH (Caesin is acidic and as it dissolves it lowers the pH).
100mM Mg.ATP
weigh in
add 1M MgAc
add cold ddH2O
add 5 M NaOH
3.086 g NaATP
5ml
40 ml
1360 µl
Add additional NaOH dropwise until pH reads 7 +/- 0.1 pH units. Adjust volume to 50 ml.
Aliquot and store at -80 – avoid refreezing after thawing.