Biology Reference
In-Depth Information
1 M DTT solution and 0.625
L IPG buffer (compatible with
the choice of pH range in IPG strip) and adjust the volume to
125
μ
L with rehydration buffer.
3. Add the sample solution into the Immobiline Rehydration tray
and gently place an Immobiline DryStrip of pH interval 4-7 of
3-11 NL onto the solution with gel-side down. Overlay the
strip with DryStrip Cover Fluid and re-swell the strip for
10-20 h.
See
Note 6
about Immobiline DryStrip and rehydra-
tion loading.
4. Move the strip to an Ettan IPGphor Isoelectric Focusing Unit
and focus the proteins using the following program: (a) step
the voltage to 300 V for 0.3 h, (b) Increase the voltage to
5,500 V by a gradient, for 0.3 h; and (c) Hold the voltage at
5,500 V until 22,000 Vh are reached. Take out the strip with
a tweezer and store at −80 °C in a 15 mL Falcon tube with
gel-side facing the internal until next step.
5. Place the strip in a 15 mL Falcon tube and add 5 mL equilibra-
tion buffer supplemented with DTT. Incubate at room tem-
perature for 15 min with rotation.
6. Replace the solution with 5 mL equilibration buffer supple-
mented with IAA and incubate for another 15 min with
rotation.
7. Place the strip with the anodic (+) end to the left and the gel side
facing forward (towards you) into a 1D SDS-PAGE gel with
one large well. Use a commercial 4-12 % acrylamide gel or
13 % acrylamide if the gel is prepared in house. Seal the gel
using the agarose sealing solution.
8. Run second dimension as described for 1D SDS-PAGE, fi x the
gel and stain with Coomassie. Pick up protein spots of
interest.
μ
1. After appropriate destaining of the gel, excise the virus protein
bands or spots of interest. Divide the bands/spots into small
pieces of approximately 1 × 1 mm and add to an Eppendorf
tube. Make sure to handle the gel on a clean surface. Use
gloves, lab coat, hair cover, and clean tools, e.g., a scalpel or
punch needle, to avoid contamination.
2. Destain the gel pieces by adding 100-200
3.3.4 In-Gel Digestion
(Suitable for Excised
Pieces from Both 1D
and 2D SDS-PAGE)
L 50 % acetonitrile,
50 % 100 mM NH
4
HCO
3
solution. Incubate at room tem-
perature in a shaker (400 rpm) for 20 min. Discard the liquid.
Repeat this step until most Coomassie stain has disappeared,
but for a maximum of two times. Add 100 % acetonitrile and
incubate for 5 min. Discard the acetonitrile and dry the gel
piece at 50 °C in for example a SpeedVac. The gel pieces will
now become white and electrostatic.
μ
