Hoefer PR625 Immunoblot - Manual (Page 17)

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Note 5. Fitting the O-rings into the slots
If your washing manifold units do not fit into
the slots easily, apply a small amount of silicone
grease around the O-rings. Place the manifold
unit loosely in the slot and press down on the
side of the manifold towards you. Then press
on the side away from you to seat the manifold
firmly in the slot.
Note 6. Secondary antibody incubations
Please follow instructions provided with your
Western blotting detection system. When
secondary antibody is used in the ImmunoBlot
at a high dilution (1:100,000 or greater), some
decrease in signal intensity may be observed.
This may be due to depletion of antibodies in
the small volume of the channel. The problem
can generally be corrected by 1) increasing the
antibody concentration 2–5 times, 2) increas-
ing the antibody volume, or 3) removing the
membrane from the unit and performing the
secondary antibody incubation in a tray.
Note: Exercise caution when
performing tray incubations
because low affinity mono-
clonal antibodies can disso-
ciate from their respective
antigens over time. Incuba-
tion in a tray permits such
antibodies to diffuse through
the incubation solution and
rebind elsewhere on the blot.
Such streaking is detect-
able as staining between
sample lanes or in negative
control lanes at the position
of one or more strongly reac-
tive bands.