Hoefer PR625 Immunoblot - Manual (Page 15)

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Step 2.
After the gels are blotted, the bands on the membrane
are not visible. Adding a non-reactive dye will make it
easy to align the membrane. This can be done in one
of two ways:
A. For larger protein loads ( > 250 ng) Ponceau S
can be used to stain the proteins reversibly on the
membrane following electrophoretic transfer.
Rinse the membrane briefly in water and stain in
0.2% Ponceau S for 1 to 2 minutes, then destain
in several changes of distilled water until red bands
appear against a white background. Since the stain
is lost during the subsequent blocking step, protein
bands should be marked at this stage with pinholes
or with a pointed pencil. The blot may also be
photocopied.
Ponceau S stained blots can be stored for months
at 4 °C, kept moist between sheets of parafilm
or buffer saturated filter paper. Blots can also be
frozen in a plastic pouch.
Ponceau S powder should be dissolved in distilled
water to make a working solution. The solution can
be reused for several weeks to months depending
upon the number of membranes stained.
B. For lower protein loads (< 250 ng), methyl green,
Pyronin Y or Deep Purple may be used to perma-
nently mark the top and bottom of the gel for subse-
quent alignment with the ImmunoBlot channels.
When loading the gel, add approximately 5 µl of
methyl green (0.1% solution in 50% glycerol) or
Pyronin Y (0.05% solution in 50% glycerol) to the
desired lanes. These dyes migrate just ahead of
the bromophenol blue dye front in the gel, and will
transfer permanently to the membrane. A second
aliquot of the dye added to the gel near the end of
the electrophoretic run will enter the resolving gel
in 5 to 10 minutes and serve to mark the top of
the gel. Please keep in mind that these dyes can
interfere with fluorescent Western blotting detection
methods and should therefore be removed prior to
imaging. If left on the membrane they will result in
nonspecific signals in fluorescent Western blotting
detection.