Biology Reference
In-Depth Information
4. After electrophoresis, gels are transferred to in-gel staining
buffers (Subheading 3.9 ).
1. Put the gel system together. Fill the upper chamber with IEF
cathode buffer and the lower chamber with IEF anode
buffer.
2. Transfer the samples in the gel pockets (Subheading 3.7 ). One
lane should be loaded with IEF-LB for pH measurements.
3. Electrophoresis is carried out over night. 12 h at 30 V, for 2 h
at 100 V, 1.5 h at 250 V and 1 h at 300 V at 4 °C.
4. pH determination: Prepare 10× 2 mL tubes and fi ll them with
1 mL water. Slice out the free gel lane only loaded with IEF-LB
and cut it in ten similar pieces (~0.6-0.7 cm). Vortex the tubes
and keep them over night at 4 °C. Keep the tubes at room
temperature for 1 h and vortex each tube for 2 min directly
before measurement with a pH-electrode. Results are used to
calculate the pH-function of the IEF-gel.
5. Transfer the rest of the gel to the according in-gel staining
solution (Subheading 3.9 ) or continue with the second
dimension.
6. Gel lane(s) for the second dimension is/are sliced out and
incubated in equilibration buffer for 45 min on a shaker. The
gel lane is transferred to the second dimension
modSDS-PAGE.
7. Fill both chambers with electrophoresis buffer. Electrophoresis
is carried out at 30 mA per gel up to 200 V at 4 °C until the
blue front is running down.
8. When the electrophoresis is fi nished the gel is transferred into
the according buffer (Subheading 3.9 ).
3.8.2 Native IEF and
Second Dimension
modSDS-PAGE
1. Put the gel system together. Fill the upper chamber with
hrCNE cathode buffer and the lower chamber with hrCNE
anode buffer.
2. Transfer the samples (Subheading 3.7 ) in the gel pockets. Add
one marker lane.
3. Electrophoresis is carried out for 30 min at 100 V, 1 h up to
500 V constricted to 10 mA per gel at 4 °C.
4. When the electrophoresis is fi nished the gel system is disas-
sembled. The part of the gel that should be used for the in-gel
staining in the fi rst dimension is sliced out and transferred into
the according buffer (Subheading 3.9 ).
5. Gel lane(s) for the second dimension is/are sliced out and
incubated in equilibration buffer for 45 min on a shaker. The
gel lane is transferred to the second dimension
modSDS-PAGE.
3.8.3 hrCNE and Second
Dimension modSDS-PAGE
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