3. Electrophoresed protein material is transferred to Hybond
ECL nitrocellulose filters for 30 min.
4. Filters are treated with 5% nonfat dry milk at +4
C for 30 min
followed by primary and secondary antibody treatment, each
for 30 min at room temperature.
5. Specific
bands
are
visualized
with
the
ECL
Chemiluminescence kit.
3.9.2 Metabolic Labeling
1. Host cells (BHK-21, CHO-K1, or HEK293) cultured on 6-,
12-, or 24-well plates are infected with serial dilutions of virus
stocks and incubate for 1–2 days at 37
C.
2. The medium is removed, cells washed once with PBS, Starvation medium added, and cells are incubated for 30 min at
37
C.
3. The medium is replaced with Starvation medium containing
50–100 μCi/mL of
35 S-methionine and cells are incubated for
20 min at 37
C.
4. The medium is removed, cells washed twice with PBS, and
Chase medium added for appropriate time (e.g., 15 min to
3 h).
5. The Chase medium is removed, cells washed once with PBS,
250 μL lysis buffer added per 6-well plate, and cells are incubated 10 min on ice.
6. Samples are loaded on 10–12% SDS-PAGE under standard
conditions, fixed in 10% acetic acid, 30% methanol for 30 min
at room temperature and replaced with Amplify
® for 30 min at
room temperature.
7. The gel is dried and exposed on Hyperfilm-MP for 2–24 h
(depending on signal) at room temperature or at À80
C
applying radioactivity-intensifying screens for visualization (see
Note 5).
3.10 Virus Stock
Purification
Although it is possible to use alphavirus particles directly for expression studies in cell lines after a single filter-sterilization procedure,
in vivo applications for immunization in animal models gain from
additional purification steps. Moreover, rigid purification procedures are mandatory for application of alphavirus particles for
clinical trials. For this purpose, various methods based on ultracentrifugation and affinity chromatography can be employed as
described below.
3.10.1
Ultracentrifugation of Virus
Stocks
1. A step gradient is prepared in ultracentrifuge tubes by addition
of 1 mL of 50% sucrose solution (bottom) and 3 mL of 20%
sucrose solution (top).
Alphavirus-Based Antigen Preparation
75
ECL nitrocellulose filters for 30 min.
4. Filters are treated with 5% nonfat dry milk at +4
C for 30 min
followed by primary and secondary antibody treatment, each
for 30 min at room temperature.
5. Specific
bands
are
visualized
with
the
ECL
Chemiluminescence kit.
3.9.2 Metabolic Labeling
1. Host cells (BHK-21, CHO-K1, or HEK293) cultured on 6-,
12-, or 24-well plates are infected with serial dilutions of virus
stocks and incubate for 1–2 days at 37
C.
2. The medium is removed, cells washed once with PBS, Starvation medium added, and cells are incubated for 30 min at
37
C.
3. The medium is replaced with Starvation medium containing
50–100 μCi/mL of
35 S-methionine and cells are incubated for
20 min at 37
C.
4. The medium is removed, cells washed twice with PBS, and
Chase medium added for appropriate time (e.g., 15 min to
3 h).
5. The Chase medium is removed, cells washed once with PBS,
250 μL lysis buffer added per 6-well plate, and cells are incubated 10 min on ice.
6. Samples are loaded on 10–12% SDS-PAGE under standard
conditions, fixed in 10% acetic acid, 30% methanol for 30 min
at room temperature and replaced with Amplify
® for 30 min at
room temperature.
7. The gel is dried and exposed on Hyperfilm-MP for 2–24 h
(depending on signal) at room temperature or at À80
C
applying radioactivity-intensifying screens for visualization (see
Note 5).
3.10 Virus Stock
Purification
Although it is possible to use alphavirus particles directly for expression studies in cell lines after a single filter-sterilization procedure,
in vivo applications for immunization in animal models gain from
additional purification steps. Moreover, rigid purification procedures are mandatory for application of alphavirus particles for
clinical trials. For this purpose, various methods based on ultracentrifugation and affinity chromatography can be employed as
described below.
3.10.1
Ultracentrifugation of Virus
Stocks
1. A step gradient is prepared in ultracentrifuge tubes by addition
of 1 mL of 50% sucrose solution (bottom) and 3 mL of 20%
sucrose solution (top).
Alphavirus-Based Antigen Preparation
75