Biology Reference
In-Depth Information
1. 200 mM HEPES buffer (10×).
2. Lysis buffer: 9 M urea, 20 mM HEPES, 2.5 mM sodium
pyrophosphate, 1 mM
2.3.2 In-Solution
Digestion
-glycerophosphate, and 1 mM sodium
vanadate. Weigh desired amount of urea and dissolve in a small
volume of water. Heat in water bath (60 °C for 5 min) and
then add a virus aliquot to this solution. Further, add small
volumes of stock solutions directly to the sample and dilute
with Milli-Q water to a fi nal volume for the desired
concentrations.
3. Trifl uoroacetic acid (TFA).
β
4. Trypsin (or other protease, e.g., LysC or chymotrypsin of
sequencing grade). Enzymes are provided as powder. Dissolve
in 1 mM HCl at a concentration of 1
μ
g/
μ
L. Aliquot 1
μ
g/vial
and store at −20 °C.
5. 10 kDa MWCO ultrafi ltration unit (Millipore or similar).
6. Empore Disks C
18
membrane (Varian).
1. Sample loading buffer for 1D SDS-PAGE (2× Laemmli sample
buffer): 62.5 mM Tris-HCl pH 6.8, 2 % sodium dodecyl sul-
fate (SDS), 25 % glycerol, 0.1 % bromophenol blue or (5×
Laemmli sample buffer): 156 mM Tris-HCl pH 6.8, 5 % SDS,
62.5 % glycerol, 0.025 % bromophenol blue. Add 1/20 or 1/8
of
2.3.3 (1D) SDS-PAGE
Separation
-mercaptoethanol (v/v) to the 2× or 5× Laemmli sample
buffer, respectively, as required.
2. Molecular weight marker: There are different markers available
provided as a powder. Store powder at 4 °C until use and then
add 2× Laemmli sample buffer with 1/20
β
-mercaptoethanol
(add the volume recommended by supplier) and heat to 95 °C
for 5 min. Allow to cool on ice and then aliquot and store at
−20 °C.
3. Running buffer: (a) If using tris-glycine gels (purchased or cast
in-house), prepare SDS-running buffer consisting of 25 mM
Tris base, 192 mM glycine, 0.1 % (w/v) SDS. Store at room
temperature. (b) If using other SDS-PAGE systems, e.g.,
NuPAGE Bis-Tris Precast gels (Life Technologies) 4-12 %,
then use the matching running buffer, e.g., NuPAGE MOPS
SDS running buffer. Store at room temperature.
4. Fixation solution: 10 % methanol, 7 % acetic acid, 83 % water,
store at room temperature.
5. Colloidial Coomassie staining, stock solution: 0.1 % CBB
(Coomassie brilliant blue)-G250, 1 % phosphoric acid,
10 % w/v ammonium sulfate in water. Store at 4 °C, add 20 %
methanol upon use.
β
1. Sample preparation solution for 2D electrophoresis: 7 M Urea,
2 M thiourea, 4 % chaps, 30 mM Tris buffer pH 8.5, 1 mM
2.3.4 (2D) SDS-PAGE
Separation
