Protein Protocols Protein Protocols

128 Walker
4. Notes
1. The sucrose is present to improve the mechanical stability of the gel. It also greatly reduces
pH gradient drift.
2. The procedure described here uses photopolymerization to form the polyacrylamide gel.
In the presence of light, riboflavin breaks down to give a free radical which initiates
polymerization (Details of acrylamide polymerization are given in the introduction to
Chapter 11). Ammonium persulphate /TEMED can be used to polymerize gels for IEF
but there is always a danger that artefactual results can be produced by persulfate oxidation
of proteins or ampholytes. Also, high levels of persulfate in the gel can cause distortion
of protein bands (see Note 10).
3. This broad pH range is generally used because it allows one to look at the totality of
proteins in a sample (but note that very basic proteins will run off the gel). However,
ampholytes are available in a number of different pH ranges (e.g., 4–6, 5–7, 4–8, and so
on) and can be used to expand the separation of proteins in a particular pH range. This is
necessary when trying to resolve proteins with very similar pI values. Using the narrower
ranges it is possible to separate proteins that differ in their pI values by as little as 0.01 of
a pH unit.
4. Ensure that your light box does not generate much heat as this can dry out the gel quite
easily by evaporation through any small gaps at the joints of the electrical insulation tape.
If your light box is a warm one, stand it on its side and stand the gels adjacent to the box.
5. It is not at all obvious when a gel has set. However, if there are any small bubbles on the
gel (these can occur particularly around the edges of the tape) polymerization can be
observed by holding the gel up to the light and observing a “halo” around the bubble. This
is caused by a region of unpolymerized acrylamide around the bubble that has been prevented
from polymerizing by oxygen in the bubble. It is often convenient to introduce a
small bubble at the end of the gel to help observe polymerization.
6. If the gel stays on the sheet of glass that does not contain the tape, discard the gel. Although
usable for electrofocusing you will invariably find that the gel comes off the glass and
rolls up into an unmanageable “scroll” during staining/destaining. To ensure that the gel
adheres to the glass plate that has the tape on it, it is often useful to siliconize (e.g., with
trimethyl silane) the upper glass plate before pouring the gel.
7. The strips must be fully wetted but must not leave a puddle of liquid when laid on the gel.
(Note that in some apparatus designs application of the electrode applies pressure to the
wicks, which can expel liquid.)
8. The filter paper must be fully wetted but should have no surplus liquid on the surface.
When loaded on the gel this can lead to puddles of liquid on the gel surface which distorts
electrophoresis in this region. The most appropriate volume depends on the absorbancy of
the filter paper being used but about 5 µL is normally appropriate. For pure proteins load
approx 0.5–1.0 µg of protein. The loading for complex mixtures will have to be done by
trial and error.
9. Theoretically, samples can be loaded anywhere between the anode or cathode. However,
if one knows approximately where the bands will focus it is best not to load the samples at
this point since this can cause some distortion of the band. Similarly, protein stability is a
consideration. For example, if a particular protein is easily denatured at acid pH values,
then cathodal application would be appropriate.
10. The most common problem with IEF is distortions in the pH gradient. This produces wavy
bands in the focused pattern. Causes are various, including poor electrical contact between
electrode and electrode strips, variations in slab thickness, uneven wetting of electrode
strips, and insufficient cooling leading to hot spots. However, the most common cause is