Detailed Protocol for Yeast Actin Purification
Jacqueline Hendries (modified 2/20/05)
I. Things to check on (at least one day before prep):
1) Have at least 3 liters of ddH20 at 4º.
2) Will need GS-3 rotor and Ti70 rotor, clean and cold.
3) Have at least 50ml deionized formamide (stored in –80).
4) Have at least 3 SDS-PAGE gels (12.5%).
II. Getting ready
1) Get two buckets of ice.
2) Begin thawing frozen reagents:
 1000x Pepstatin (liquid, stored in -20°C)
 AqPI (liquid, stored in -20°C)
 1M DTT (liquid, stored in -20°C)
 ATP (solid, stored in -20°C)
 Deionized formamide (liquid, stored in -80º)
 Keep Pepstatin and ATP on ice, let DTT and AqPI thaw at RT.
 Make sure you have 0.2M PMSF stock (stored at room temperature)
3) Set up centrifuges

Sorvall to 4º, put in GS-3 rotor

Beckman LE-80 Ultra-centrifuge to 4º.
4) Make 1L of G-buffer fresh at room temperature.
III. Preparing the yeast lysate/supernatant
1) You will need:
 2L glass beaker
 metal spoon
 2 ice buckets about half full of warm tap water
 2-4 GS-3 rotor bottles, 5ml pipet and autopipetter set near centrifuge scales
 1000l and 200l PipetMen, set to add PMSF and AqPI/Pepstatin (167µl and
34µl respectively, per 100g yeast)
2) Weigh 100g (or your weight) frozen yeast powder directly into a 2L glass beaker.
Use spoon to scoop.
3) Add an equivalent amount of G-buffer to your yeast and stir – add the G-buffer in
two steps.
 Accelerate the melting by setting the glass beaker into the bucket of warm
water. When the water gets cold, switch to the other bucket.
4) Add AqPI, Pepstatin and PMSF in three steps – when the yeast first gets mushy
(icy chunks), when the yeast is about half chunks and half liquid, and when the
yeast is almost all liquid.
5) When the yeast is fully melted, transfer to GS-3 rotor bottles.
6) Spin to make LSS – 12K rpm for 15-20 minutes. While it’s spinning…

Set up a 250ml graduated cylinder, funnel and 2 pieces of cheesecloth (in
funnel) to collect LSS, also 25ml disposable pipet and Kimwipes

Filter sterilize the rest of your G-buffer (used filter -> glass bottle) and put in
FPLC cabinet to chill

Make another 1L of G-buffer “cold,” using cold ddH2O from the cold room

Run cold G-buffer through DnaseI column(s) and adjust flow rate to 1-2
ml/min (per column)

Chill tubes/caps for Ti70 rotor and put on ice in centrifuge room.

Remember to go back and check your columns! DO NOT let them go dry.
7) Work in cold room at this point. Pour LSS through cheesecloth and into
funnel/cylinder.

Volume of LSS: ________________ ml
8) Aliquot about 26.5ml of LSS into each of the Ti70 bottles and keep on ice.
Tighten cap and check that there is not too much air in the tube.
 If you do not have exactly even amount of LSS, just leave the bottom few ml.
If it’s very close (>20ml) adjust up with cold G-buffer.
9) Wipe the bottom of the bottles and put into Ti70 rotor.
10) Spin to make HSS – 60Krpm, 80 minutes (1:20). While this is going…

Adjust DnaseI columns/flowrate if you still need to (this can take a while).

Set up for collecting HSS (centrifuge bench is a good spot)
 2L plastic beaker for “trash”
 250 or 500ml flask on ice, funnel and 2 cheesecloth
 tweezers to slide bottles out of rotor
 2 x 10ml disposable pipets and old autopipetter

Make buffers for DnaseI and MonoQ (store cold)
G-buffer
Other
 G + 10% formamide 45ml G buffer
5ml d.i.formamide
 G + 0.2M NH4Cl
50ml G buffer
0.535g NH4Cl
 G + 50% formamide 25ml G buffer
25ml d.i.formamide
 G + 1M KCl (fs)
300ml G buffer
22.4g KCl

Set up for DnaseI eluate – 20 eppie tubes numbered sequentially; filter paper
with 1-20 grid, 20 eppies with 5l sample buffer each.
 Vol.
50ml
50ml
50ml
300ml
11) Harvesting the HSS

Be very very careful while moving and opening rotor!

Carefully slide bottle out of rotor with tweezers

Carefully unscrew cap; pull out plastic plug as smoothly as possible,

Hold the bottle as steadily as you can while you pipet HSS out:
 Dunk pipet tip under the white lipid layer and pipet up about 12ml,
transfer to cheesecloth/flask on ice.
 “Presuck” droplets in pipet to avoid disrupting surface when you go in for
remaining HSS.
 Suck up another 5-7 ml or so. Don’t be too ambitious…
 Attempt another 1-2ml, but under no circumstances suck up the crud at
the bottom!
 Discard bottle and cap in 2L beaker. Repeat as needed!
(a) Volume HSS ________________ ml (approx.)
The DnaseI column
12) Load the HSS onto the column at a rate of about 1-2ml/min. Calculate how long
this will take (based on volume of HSS): ________________
 Be paranoid!! Tape the inlet tubes to the HSS flask and check frequently that
they have not slipped.
 For last 10-15ml or so, transfer to a 15ml falcon so you can get (almost) every
last drop and avoid air bubbles.
 Take a sample of the flowthrough midway through (50l), for troubleshooting
gel/blot. Add to 25µl of 3x sample buffer and boil 3’.
 Also save a small amount of HSS – same procedure as for flowthrough.
 While working with the DnaseI column…
13) Prepare the MonoQ

Wash the pump and columns with water
 Pumpwash: A on, B on, gradient 0%B (execute)
 When pumpwash is done, set flow to 0.5ml/min. Run for 20min (10c.v. of
water)

Wash the pump with G-buffer
 Same as last steps, but inlet tube in G-buffer instead of H2O
 Repeat with high salt (100%B)
 Repeat once more with G-buffer (0%B)

Wash loop (ensure system is set at Load; inject 3 x 5ml G-buffer into 5ml
loop)

Takes about 1.5 hr to clean thoroughly but is pretty low maintenance.
14) Get concentration stuff ready – get out concentration falcon, balance tube, etc.
Also set up for running a protein gel.
15) Washing DnaseI column (take a 50l sample of flowthrough at each step). Stop
the machine when you switch buffers!

2 c.v. G-buffer

2 c.v. G + 10% deionized formamide

2 c.v. G + 0.2M NH4Cl

2 c.v. G-buffer

Take a sample of each wash (50µl + 25µl sample buffer, boil 3’)
_______________ ml
____________ min
16) Eluting from DnaseI

2 c.v. G + 50% deionized formamide

Take 1ml fractions from the time you start the formamide. At fraction #20,
stop and do a Coomassie filter paper test (2µl each fraction on your labeled
Whatman filter paper, soak in Coomassie and rinse with water). Continue
collecting fractions up to 25 if 20 spot is dark.

Run a 12.5% Coomassie gel on fractions and pool those with most actin. You
can base the pooling on the filter paper test but run the gel just to check

Pooled fraction #s: _______________________
Do final check on MonoQ (program, fraction collector, etc) and get needle, syringe
ready to inject sample.
17) Inject sample onto MonoQ using 5ml loop. If you have >5ml actin, inject first
5ml and allow to load, then inject the rest of your sample (make sure the program
will allow you to do this).

Set up for 2 more gels (process and MonoQ): 14 tubes with 10l sample
buffer each.

Prepare for dot blot (mark membrane)

Wash DnaseI column(s) with copious amounts of G-buffer. Save the
flowthrough, just in case.
18) Run gel on peak fractions (2l each) Fractions run: _____________
19) Simultaneously, dot blot for cofilin (2l) – use a positive control NOT in SDS
buffer!
 Block 2’ with skim milk in TBST (prepare cofilin Ab)
 20’ incubation with 1º Ab (cofilin) in skim milk (prepare chicken Ab)
 3x rinse with TBST
 20’ incubation with 2º Ab (anti-chicken) in TBST (prepare exposure stuff)
 3x rinse with TBST
 2 x 5’ wash with TBST

Expose 1’ with commercial ECL reagent
20) Use dot blot to quantitate cofilin (by eye) and decide which fractions to pool:

Pool “pure”: _______________
w/cofilin: ______________
21) Polymerize:
 Add fractions to 0.5mM ATP and 2mM MgCl2 (0.5µl 2M MgCl2 and 2.5µl
0.1M ATP per fraction pooled) in a TLA 100.3 tube. ADD THE
FRACTIONS TO THE MIX, not the other way around.
 Mix gently with your pipet (don’t worry about shearing at this point).
 Parafilm the top of the tubes and leave at room temp for 20 minutes.
22) Leave at 4º overnight! (You can continue the prep tonight if you wish: let
polymerize for 1 hour at room temp and continue at step VI. Check the “Before
you leave” stuff while polymerizing.)
IV. Before you leave…
1) Make sure the MonoQ is clean (washed with 1M KCl, then G, then water, then
20%EtOH) and OFF.
2) Make sure your DnaseI columns have been cleaned thoroughly (about 10c.v. of G
buffer is sufficient). If you will not be using them again soon, wash them into G
buffer + azide.
3) Clean up after yourself.
4) Consolidate your gels – you have at least three – and other diagnostics, i.e., filter
paper, dot blot, etc. Put the gels in one big Coomassie container or, if they’ve
destained enough, dry them.
5) Check to make sure you’re ready for tomorrow:
 Proper size of tubing (MWCO<10,000)
 Sign up for the TLA centrifuge
 Clean dounce apparatus (usually you will use the 2ml, but if you have a very
large prep you may want to try a larger one).
V. Day II and beyond
1) Take the TLA 100.3 rotor and your actin out of the cold room and let sit at room
temp for a little while.
2) Add 3M KCl (to 600mM – add 1/4 volume of pooled actin for 1/5 total volume)
to the cofilin containing fractions – this helps separate the actin from the cofilin.
Mix with a cut pipet tip.
3) Centrifuge 90Krpm, 60 min. at 20ºC in high speed centrifuge
4) While centrifuging, prepare for dounce and dialysis:

Calculate how much G-buffer to add to the actin to get 1-2mg/ml final
concentration (based on MonoQ gel):

Pure: _________________
With cofilin: _____________

Soak dialysis tubing in G-buffer (1-2L beaker) in 4º room.

Set up for douncing – get 200µl PipetMan, glass Pasteur pipets, small bulb,
dounce apparatus.

Just before the actin is done spinning, set up your dialysis tubing – clip one
end, bring buffer out to your bench.
5) Remove actin from centrifuge, carefully pipet off supernatant and save a 50µl
sample (same method as before).
6) Resuspend the clear actin pellet in G-buffer. Add in small amounts to make
resuspension easier. It is okay to use the pipet tip to scrape the actin off the sides
of the centrifuge tube.
7) Transfer the resuspended liquid into your dounce apparatus, again in small
amounts.
8) Dounce until pieces of actin are no longer visible. Be careful not to get too many
air bubbles into the dounce. Twisting the pestle gently can make the process go
more quickly.
9) Use a Pasteur pipet to transfer the actin, in small volumes, to your dialysis tubing.
Clip off the tubing (it is okay if you get an air bubble here, it is preferable to have
a little air than to lose your actin) and place it in the G-buffer.
10) Allow to dialyze overnight at 4°C (in cold room)
11) Next day – replace your dialysis buffer with fresh G-buffer. Dialyze overnight
again.
12) Check your concentration by taking OD (use the quartz cuvette): 38.5 µM *
OD290
13) Aliquot 10 or 20µl aliquots in small tubes and snap freeze (in liquid nitrogen).
Store at -80º.
14) Run a quantification gel with one of the aliquots and known standards of actin and
cofilin; calculate mg/ml and note this on the aliquot box:
Pure: _______________mg/ml
with cofilin: _______________mg/ml