Bio-Rad Model 111 Mini IEF Cell - Manual (Page 20)

6.3 Sample Preparation
Protein samples for isoelectric focusing must be free of precipitates. Substantially salt-free
samples in typical biochemical buffers are usually tolerated, though better results can be obtained
with solutions in deionized water, 2% ampholytes, or 1% glycine. Suitable protein solutions may
be prepared by dialysis, or gel filtration with Bio-Gel P-6DG desalting gel (catalog number 150-
0738).
A convenient technique for preparing small samples is to load a small amount of hydrated
Bio-Gel P-6DG gel into a 0.5 ml plastic microcentrifuge tube with a small hole in the bottom.
Load the sample on top of the gel and place this tube into a 1.5 ml plastic microcentrifuge tube.
Spin for 5 seconds in a microcentrifuge. The sample will be adequately desalted for isoelectric
focusing, and can be isolated from the bottom of the larger tube.
Many samples will require the use of urea, ethylene glycol, non-ionic detergents (i.e. Triton
X-100, NP-40, Lubrol WX, or octylglucopyranoside), or zwitterionic detergents (CHAPS,
CHAPSO). Even in the presence of detergents, some samples may resist solubility due to salt
requirements. Only if high salt is an absolute requirement should it be present in a sample, and
even then, substantial distortions and anomalies can be expected.
6.4 Sample Application
Sample application is most conveniently accomplished using the included Sample Templates.
1. Place the template on top of the polymerized gel. The colored portion of the template should
coincide with the longer dimension of the gel. The application position for the sample varies
(see Section 6.5) but one should allow 1 cm at both the top and bottom of the gel where the
gel will contact the electrodes.
2. Apply samples using a pipettor capable of delivering between 0.5 µl and 2 µl. Volumes above
2 µl are not generally recommended when using the sample template.
3. Allow samples to diffuse into the gel for 5 minutes.
4. Carefully remove the template from the gel.
Note: For larger amounts and sizes of samples, one may custom-form application strips from
filter paper, or use Bio-Rad’s Sample Application Pieces (catalog number 170-4257). Apply
the sample to the filter paper and then place the filter paper at the point of application on the
gel. Allow the sample to diffuse into the gel for 5 minutes. The disadvantage of this method is
that some proteins may not be completely eluted from the strip to the gel.
6.5 Position of Application
There are no fixed rules regarding the positioning of the sample on the gel. Samples should
not, in general, be applied in areas where protein bands are expected to focus. To protect the
proteins from extreme pH exposure, the samples should not be applied closer than 1 cm from
either electrode. The best strategy for a new protein is to make three points of application, one at
each end and one near the middle of the gel, and observe the resulting focusing pattern.
6.6 Set Up Procedure
1. Slide the lid of the Model 111 Mini IEF Cell toward the electrode plugs to remove it.
2. Remove the Graphite Electrodes from the cell and rinse them with distilled water to remove
traces of agarose from the previous run. The electrodes may be gently wiped with a laboratory
tissue if necessary. Place the electrodes back into the cell.
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