Mark Dayel
11/15/01
The night before:
Bind the N-WASP to the CH-Sepharose, and check that the
DEAE is cycled.
To bind N-WASP:
Rationale: The N-WASP binds to NHS on the resin via the primary amines on
the lysines and the N-terminus. For the
binding to occur, there must be absolutely no other primary amines in the
solution i.e. no Tris, no Azide. DTT
also blocks binding. We add high salt
to unfold the protein, prevent aggregation, and expose the lysines. Coupling occurs at pH 5—10. Increasing pH inactivates the resin by
causing hydrolysis of the NHS, but increases the coupling reaction rate by
deprotonating the –NH2’s. Therefore we
willl couple first at low pH, then bring the pH up to 8 by adding HEPES to
couple any remaining protein.
Thaw a tube of 1mL 1mM N-WASP from the
-80. Add 1ml cold water, transfer to a
50mL conical and add KCl to 500mM and dissolve.
Pour ~200ml cold water into a beaker
and pH to 2.0 with conc HCl.
Weigh out 1g of dry CH-Sepharose into
a 15ml conical.
Carefully pour in about 8ml of the pH2
water and rock to swell the resin. Take
care to get all the resin wet---it tends to get stuck at the end when it
swells. Rock in cold for 5 mins.
Set up the small glass scintered
funnel under vacuum. There’s a Wheaton Homogeniser plunger that just fits
snugly in—get this ready. Pour in the
resin and wash with all the pH2 water.
Don’t let it go completely dry.
Take off the top of the funnel—the
resin should stay in. Hold it over the
50mL conical with the N-Wasp, and push the resin into the protein with the
little plunger from the Wheaton Homogeniser.
Rinse off the funnel before the resin dries on.
Gently rock the N-WASP ovenight in the
cold room. The following day, add 200uL
of 1M HEPES pH8.0, and rock at room temperature for 2 hours. Block by adding ~1mL of 1M ethanolamine and
rocking at room temp for 1hour.
On the day of the prep:
Get up early.
Come in and take out four (4) of the 20g amoeba tubes from the -80. Make up 200ml extraction buffer:
~160ml cold water
22g sucrose
4ml 1M Tris pH8.0 @4°C
5mM DTT
1mM ATP
2mL 100mM EGTA
2ml 100x protease inhibitor cocktail
2ml 200mM PMSF
0.1mg Benzamadine
Check that pH is 8.0, pour 20ml of extraction buffer
into each tube and melt the amoebas.
Break up the amoeba pellet with one of the big metal stirrer bars—this helps
it melts very quickly. Once melted,
keep on ice. Use the entire extraction
buffer.
Use the overhead stirrer in the Agard lab cold room and
the Wheaton Homogeniser to lyse the cells.
Turn the dial to about 3–4, and dounce 4 times.
Spin in the GSA rotor at 13,000 RPM for 20 minutes, take
the supernatant and spin at 38,0000 RPM in the Ti50.2 for 90 minutes. In the meantime...
Make up the column buffer (2 litres):
2mM Tris, pH 8.0
1mM DTT
200mM CaCl2
1mM ATP
Pour and start equilibrating a column with 15mL of C200
resin and the NWASP column with column buffer (100mL)
Pour ~100mL cycled DEAE into the little Buchner funnel
and equilibrate in column buffer (300mL)
Once the Ti50.2 rotor spins down, carefully take out the
tubes and use a pippetteman with a 10mL pipette to remove the cytosol beneath
the layer of lipid. The lipids form a
layer ~5mM thick at the top. Also be
careful not to disturb and suck up any of the pellet. Pipette the cytosol into a 300mL beaker and stir with 45mL of the
equilibrated DEAE resin. Put the remaining 5ml of DEAE into the bottom of a
100mL blue column. Batch bind the DEAE
to the high speed sup, stirring for 20 minutes.
Pour the DEAE into the 100mL blue column and collect the
flow through. Once the flow stops, pour
in 100mL column buffer on top and collect the flow through.
Put this flow through over the C200 column and collect
the flow through again. The top of the
C200 column should go pink.
Add 25mM KCl to the flow through, and pass over the
N-WASP column. Discard the flow
through.
Meanwhile make the wash and elution buffers:
Wash 1: 20ml column buffer + 25mM KCl.
Wash 2: 20ml column buffer + 100mM KCl.
Elution: 50ml column buffer + 400mM MgCl2.
Wash the NWASP column with Wash1, then Wash2. Meanwhile equilibrate 300mL of low sub phenyl-sepharose resin in elution buffer and make up 1 Litre
of 1xKMEI + 1mM TCEP + 100mM ATP. Equilibrate a PD-10 column with 25mL of this KMEI. Keep the rest to dialyse the protein against
after the sucrose concentration at the end.
Set up a row of eppendorf tubes in one of the metal
blocks in an ice bucket. Carefully
elute the N-WASP column with elution buffer collecting around 10, 1mL
fractions, and measure A290 using the spec. [Arp2/3] = A290 * 7.14
Pool the peak fractions and put over the phenyl
sepharose column. Add 300mL of elution buffer to chase.
Collect the flow-through, and chase with 300mL elution buffer.
Load this flow-through onto the PD-10 column (max 3ml at
a time) and collect the salt-exchanged fractions. The protein comes out quickly—take 0.5mL fractions and it comes
out usually in the first 8. Measure
A290s on the spec, pool the peak fractions.
That’s it, unless you neet to concentrate it, in which
case dialyse vs sucrose for a bit (1–2 hours), then pop it back into the KMEI,
and use the next day.
Cycle the C200 by washing in the column with ~100mL
saturated KCl and then with Tris buffer.
Cycle the DEAE as usual with KOH and HCl.