Protein Protocols Protein Protocols

Ultra-Thin Polyacrylamide Gels 129
excessive salt in the sample. If necessary, therefore, samples should be desalted by gel
filtration or dialysis before running the gel.
11. Although not absolutely essential, removal of sample strips at this stage is encouraged
since bands that focus in the region of these strips can be distorted if strips are not removed.
Take care not to make a hole in the gel when removing the strips. Use blunt tweezers
(forceps) rather than pointed ones. When originally loading the samples it can be advantageous
to leave one corner of the filter strip slightly raised from the surface to facilitate
later removal with tweezers.
12. It is indeed good idea to include two or three blood samples in any run to act as markers
and to confirm that electrophoresis is proceeding satisfactorily. Samples should be prepared
by diluting a drop of blood approx 1:100 with distilled water to effect lysis of the
erythrocytes. This solution should be pale cherry in color. During electrophoresis, the red
hemoglobin will be seen to electrophorese off the filter paper into the gel and ultimately
focus in the central region of the gel (pH 3.5–10 range). If samples are loaded from each
end of the gel, when they have both focused in the sample place in the middle of the gel
one can be fairly certain that isoelectrofocusing is occurring and indeed that the run is
probably complete.
13. Theoretically, when the gel is fully focused, there should be no charged species to carry a
current in the gel. In practice there is always a slow drift of buffer in the gel (electroendomosis)
resulting in a small (~0.5 mA) current even when gels are fully focused. Blood
samples (see Note 9) loaded as markers can provide additional confirmation that focusing
is completed.
14. It is not possible to stain the IEF gel with protein stain immediately following electrophoresis
since the ampholytes will stain giving a uniformly blue gel. The fixing step allows
the separated proteins to be precipitated in the gel, while washing out the still soluble
ampholytes.
15. This brief wash is important. If stain is added to the gel still wet with fixing solution, a
certain amount of protein stain will precipitate out. This brief washing step prevents this.
16. If you wish to determine the isoelectric point of a protein in a sample, then the easiest way
is to run a mixture of proteins of known pI in an adjacent track (such mixtures are commercially
available). Some commercially available kits comprise totally colored compounds
that also allows one to monitor the focusing as it occurs. However, it is just as easy
to prepare ones own mixture from individual purified proteins. When stained, plot a graph
of protein pI vs distance from an electrode to give a calibration graph. The distance moved
by the unknown protein is also measured and its pI read from the graph. Alternatively, a
blank track can be left adjacent to the sample. This is cut out prior to staining the gel and
cut into 1 mm slices. Each slice is then homogenized in 1 mL of water and the pH of
the resultant solution measured with a micro electrode. In this way a pH vs distance
calibration graph is again produced.