Proteome Analysis of Adenovirus Using Mass Spectrometry - Adenovirus: Methods and Protocols - page 36

For troubleshooting, see Note 3 about how to remove
detergents, Note 4 on how to check sample quality and Note
5 for control of digestion effi ciency.

1. Take out desired volume/amount of purifi ed virus, e.g., 25 μ L

3.3.2 (1D) SDS-PAGE
Separation

corresponding to 12-25 μ g, and aim for a total volume of

100 μ L. Add 20 μ L of 5× Laemmli sample loading buffer,

10 L of Milli-Q water (adjust the

water volume depending on what volume of virus is used).
Incubate at 95 °C for 5 min. Cool until room temperature and
then add 10

μ L of 45 mM DTT, and 35 μ

μ L of 100 mM IAA. The total volume is now

100 L. Incubate 5 min in the dark at room temperature

(if less than 100

μ

L of sample is needed, divide the volumes, or
for concentrated samples, replace water with virus sample).
The sample is now reduced and alkylated.
2. Analyze the sample on a 1D SDS-PAGE known to resolve all
expected protein bands. Gels can be purchased pre-cast or
produced in-house following generally available protocols.
We recommend a 13 % gel to resolve and detect the proteins in
the Ad2 virus capsid. Add the sample together with a low
molecular weight marker and run the gel with settings suitable
for your type of electrophoresis system and gel type. Stop the
gel when the front migrates out to make sure that low molecu-
lar proteins are not lost.
3. Take out the gel and treat it with fi xation solution. Incubate
for 45 min to 1 h.
4. Mix methanol and Coomassie staining stock solution 1:4 and
incubate overnight at room temperature. Destain the gel by
washing several times with water.

μ

1. Protein extraction using 2D electrophoresis: Take out a volume
of virus solution corresponding to around 500

3.3.3 (2D) SDS-PAGE
Separation