Choice of electrophoretic
conditions for peptide mapping
Traditionally, 35S-methionine-labeled proteins have been mapped
at pH 4.72 and 32P-labeled proteins at pH 8.90. Some people favor
pH 1.9 for both. There are no rules. Use whatever pH gives a
useful distribution of peptides. However, an issue to keep in
mind is that it is valuable if your maps can be compared easily
with those of other people who are mapping the same protein.
If everyone else in the field is using one pH, it's sensible
to use that pH too.
and 4.72 maps. The origin
in these maps is in the lower left, up 2.5 or 3 cm from the bottom
and in 5 cm from the left edge of the plate and the positive
electrode is on the left. At these acidic pHs, most peptides
are postively charged and therefore migrate toward the minus
Electrophoresis is done for 27 min at 1 kV using the MBVL rigs
or the CBS rig.
maps. The origin here is in the middle of the plate, up 3 cm
from the bottom. These maps are sometimes run with the origin
placed at the back of the rig. There has been occasional trouble
with puddling of buffer in 8.9 maps. Since the origin is in the
middle of the plate, it doesn't matter which side the anode is
Again, electrophoresis is for 27 min at 1 kV on an MBVL rig.
Tony likes to do the electrophoresis for 20 min. This gives less
separation, but keeps the free phosphate on the plate.
Note that these electrophoresis times are
correct only for an MBVL-type rig. If another rig is used, it
is likely that a different number of volt/minutes will have to
be used. These should be determined empirically.
We have two kinds of chromo buffer.
Regular was developed
by Wade Gibson for iodinated peptides. It is useful for hydrophobic
peptides, such as those containing the amino acid labeled during
The 32P or phosphochromo
buffer was developed by Nancy Axelrod. It is less hydrophobic
and gives better separation of non-hydrophobic peptides such
as those containing phosphorylated amino acids.
Additionally, a third buffer containing, among other things,
isobutyric acid, is currently in vogue.
You should use the buffer that gives the best separation of the
peptides you are interested in.
Traditionally, regular buffer is used for maps of 35S-methionine
labeled proteins and phospho buffer for phosphorylated peptides.
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