11. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE).
(a) Sample treatment (both, protein alone, and the conjugation mixture).
l
Use 5–20 μL of the samples with a protein content of
5–10 μg.
l
Mix the samples with 0.5 M dithiothreitol solution
(1/5 v/v) and NuPAGE LDS sample buffer (1/5 v/
v).
l
Spin them in the centrifuge in order to draw down all
the volume in the bottom of the tube.
l
Place the tubes in a 100
C preheated block heater
and boil the samples for 1 min.
l
Spin again all samples in the centrifuge in order to
draw down all the volume in the bottom of the tube.
(b) Buffer preparation and system setup.
l
Use a NuPAGE
® Novex 7% Tris-Acetate Gel (see
Note 29).
l
Dilute 25 mL of Tris-Acetate running buffer 20Â
with 475 mL of water in a graduate glass cylinder
and transfer into the middle part of the cell up to
cover the gel wells and more up to 2 cm from the
front bottom of the cell.
l
Insert the gel in the electrophoresis cell after having
removed the strip present in the bottom of the gel.
(c) Sample loading.
l
Remove the wells protection from the gel.
l
Use the first well to load few microliters of an appropriate marker.
l
Place all the samples in the wells noting the
lanes used.
(d) Electrophoresis run.
l
Close the cell and connect it to the power supply.
l
Set up the power supply at 200 V and 45 mA and run
the gel.
l
Wait until the blue marker of the wells reaches the
bottom of the gel.
(e) Gel Staining.
l
Open the cell and remove the gel from its holder
using the gel knife.
O-Antigen Purification and Conjugation
297
(SDS-PAGE).
(a) Sample treatment (both, protein alone, and the conjugation mixture).
l
Use 5–20 μL of the samples with a protein content of
5–10 μg.
l
Mix the samples with 0.5 M dithiothreitol solution
(1/5 v/v) and NuPAGE LDS sample buffer (1/5 v/
v).
l
Spin them in the centrifuge in order to draw down all
the volume in the bottom of the tube.
l
Place the tubes in a 100
C preheated block heater
and boil the samples for 1 min.
l
Spin again all samples in the centrifuge in order to
draw down all the volume in the bottom of the tube.
(b) Buffer preparation and system setup.
l
Use a NuPAGE
® Novex 7% Tris-Acetate Gel (see
Note 29).
l
Dilute 25 mL of Tris-Acetate running buffer 20Â
with 475 mL of water in a graduate glass cylinder
and transfer into the middle part of the cell up to
cover the gel wells and more up to 2 cm from the
front bottom of the cell.
l
Insert the gel in the electrophoresis cell after having
removed the strip present in the bottom of the gel.
(c) Sample loading.
l
Remove the wells protection from the gel.
l
Use the first well to load few microliters of an appropriate marker.
l
Place all the samples in the wells noting the
lanes used.
(d) Electrophoresis run.
l
Close the cell and connect it to the power supply.
l
Set up the power supply at 200 V and 45 mA and run
the gel.
l
Wait until the blue marker of the wells reaches the
bottom of the gel.
(e) Gel Staining.
l
Open the cell and remove the gel from its holder
using the gel knife.
O-Antigen Purification and Conjugation
297