Biology Reference
In-Depth Information
2.2 Virus Purifi cation
1. Storage buffer for virus: 10 mM Tris-HCl, pH 7.9. Prepare
this solution fresh as required by diluting from a stock solution
of 1 M.
2. Purifi cation reagents: 5 % (w/v) sodium deoxycholate, make
fresh as required.
3. CsCl solution of density 1.41 g/cm 3 : Weight 27.42 g CsCl
and fi ll to 50 mL with 10 mM Tris-HCl pH 7.9. Sterile fi ltrate
the CsCl solution by 0.22
m fi lters. Densities can be double-
checked by weighing. All CsCl solutions are long term stable
in room temperature.
4. CsCl solution of density 1.34 g/cm 3 : Weight 22.71 g CsCl
and fi ll to 50 mL with 10 mM Tris-HCl pH 7.9. Sterile fi ltrate
the CsCl solution by 0.22
μ
m fi lters.
5. CsCl solution of density 1.27 g/cm 3 : Weight 18.47 g CsCl
and fi ll to 50 mL with 10 mM Tris-HCl pH 7.9. Sterile fi ltrate
the CsCl solution by 0.22
μ
m fi lters.
6. For dialysis: Slide-A-Lyzer Cassette (10 kDa MWCO, 0.5-3 or
3-12 mL, Pierce/Thermo Scientifi c Inc.) and 10 mM Tris-
HCl, pH 8.0.
7. Ultra-clear ultracentrifugation tubes (344058, Beckman Coulter).
8. AH-429 rotor and ultracentrifuge.
μ
1. Phosphatase inhibitors can be prepared from the following
stock solutions:
(a) 500 mM NaF (500×), prepare in water, aliquot and store
in freezer.
(b) 1 M
2.3 Preparation of
Samples for Analysis
of the Viral Proteome
by Mass Spectrometry
-glycerophosphate (500×), prepare in water, aliquot
and store at −20 °C. Thawed aliquots can be stored at
4 °C for 6 months.
(c) 500 mM Na-pyrophosphate (500×), prepare in water,
store at 4 °C for up to 1 month.
(d) 500 mM Na 3 VO 4 (500×) needs to be activated/depoly-
merized. Add powder and fi ll to 90 % of desired volume
with water. Adjust pH to 10 using 1 M HCl. At this pH
the solution becomes yellow. Boil the solution until it is
colorless and put on ice for cooling. Readjust the pH to 10
and repeat boiling and cooling until the solution remains
stably colorless at pH 10. Adjust to desired volume with
water, aliquot and store at −20 °C.
2. 1-1.25 M dithiothreitol (DTT) Aliquot and store at −20 °C.
3. Iodacetoamide (IAA): Purchase as powder, weigh desired
amount each time a solution should be prepared in Milli-Q water.
4. Acetonitrile.
5. EZQ protein quantitation kit (Molecular probes) or similar.
β
2.3.1 Common
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