cytiva Ettan IPGphor Cup Loading Manifold User Manual page 19

Sample application (loading)
Follow the instructions below to load the sample.
Step
1
2
3
Paper-bridge loading
Large sample volumes and large protein amounts can be applied using paper bridge
loading. A paper pad (paper bridge) is soaked with sample and placed between the
anodic end of the Immobiline DryStrip gel and the electrode 375– 500 μl sample can be
applied using the paper bridge pads.
Solutions containing up to 5 mg protein have been loaded on a 18 cm-long narrow pH
range Immobiline DryStrip gel. The rehydrated Immobiline DryStrip gel is first posi-
tioned in the bottom of the manifold channel. Then the paper bridge with sample is
positioned, followed by a paper wick (see following figure).
With anodic application the anode electrode is positioned as far out as possible in the
electrode assembly, while the cathode electrode is positioned close to the end of the
Immobiline DryStrip gel to ensure good contact between the paper wick and Immobi-
line DryStrip gel.
Note:
Ettan IPGphor User Manual 11003460 AE
Action
Briefly centrifuge the protein sample prior to loading to remove insoluble
material and particulate matter. These materials will impede sample entry
and result in vertical streaks in the second-dimension gel.
Load samples into the sample cups. A maximum of 150 μl of sample may be
placed in these cups. Check to make sure that there is cover fluid over the
samples. When the cups are initially placed on the manifold, cover fluid will
flow into the cups as they are seated. When sample is introduced into the
cups, the sample will sink to the bottom of the cup and contact the IPG strip.
Note:
For basic IPG strips, superior focusing patterns are generally obtained when
the sample cup is placed at the anodic (+) end of the Immobiline Dry Strip.
Close the Ettan IPGphor lid.
The anodic application point is an important factor for obtaining good
results.
3 Operation
3.1 Instructions
19