Biomedical Engineering Reference
In-Depth Information
Table 2.11.
Running conditions for IPG gels
Sample in applicator
Sample applied during rehydration
1 h 150 V
1 h 150 V
2 h 300 V
2 h 300 V
1 h 600 V
8 h 3500 V
8 h 3500 V
Marker proteins with known isoelectric point should be used to
check the pH gradient. These proteins are stained by the common
protocols (e.g., Coomassie Brilliant Blue, Protocol 2.3.1.2, or silver
staining, Protocol 2.3.2).
If the IPG gels are not used for a second dimension run, the gels
are further processed (stained, blotted) as described for SDS gels.
2.1.11.2 Second Dimension: SDS-PAGE
(Acrylamide Gradient Gel)
The discontinuous Laemmli SDS-PAGE with polyacrylamide con-
centration gradient in the separating gel is mostly used for second
dimension in 2D-PAGE (cf. Protocol 2.1.1). Of course, other elec-
trophoretic systems are also applicable.
For convenience, prepare the gel in vertical position but run
the second dimension on a horizontal apparatus.
Form a cassette by glass plates 1 - 2 cm wider than the length of
the IPG strip and with 0.75 to 1 mm thick spacers. Cover one plate
with a thoroughly water-washed GelBond PAGE sheet (hydrophilic
surface to the lumen of the cassette).
Prepare the gel as described in Protocol 2.1.1, i.e., first a sep-
aration gel followed by a stacking gel without sample application
slots.
E6Murea,30%glycerol(w/v),2%SDS,50mMTris-HCl, pH 8.8,
Solutions/Reagents
/
ml bromophenol blue in ddH
2
O
E
0.2 M iodoacetamide in buffer E
FirstequilibratetheIPGstripfor15min in sufficient buffer E, then
for an additional 15 min in buffer E
. Rinse the strip with ddH
2
O
after equilibration and suck off carefully remaining water by filter
paper.
Place the IPG strip face down onto the stacking gel, about 5 mM
apart of the cathode paper bridge. Place silicone rubber sample
applicators for molar mass marker proteins at one or both sides of
the IPG strip.
Run the electrophoresis as described in Protocol 2.1.1. Cool
the gel to about 15
◦
C during the run. When the tracking dye
bromophenol blue has reached the interphase between stacking
and separation gel, stop electrophoresis. Remove the IPG strip and
move the electrode paper strip to that place where the IPG strip
0.125 mg
