Hoefer PR625 Immunoblot - Manual (Page 10)

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Leaking – antibody
solution has moved to
adjacent channels. Dry areas of the membrane act as
wicks for fluid from adjacent areas. Always pre-wet the membrane before
mounting in the unit.
Fill every channel, covering the
membrane with buffer.
Membrane did not cover the full
length of the channel. Ensure that membrane is cut to
proper size.
Fluid in overloaded channels spilled
out when unit was rocked causing
contamination of adjacent channels. Do not overfill channels.
Re-used sealing pads did not seal
properly. Do not re-use sealing pads as they
compress during use.
Tray incubation. Consider incubating your blot with
secondary antibody in the ImmunoBlot
rather than tray incubation. (See
Appendix A: Note 6.)
Membrane is stripped of
blocking protein. Please take care not to strip the blot of
blocking protein while washing it prior
to addition of primary antibodies. Please
follow instructions from your Western
blotting detection kit. A sample protocol
is given in Appendix B.
Unwashed channels contained
antibodies that contaminated the
experiment. Clean the unit after every use. For
cleaning tips, see the Clean the
ImmunoBlot section, on page 7.
Low Sensitivity When low affinity or low titer
antibodies are used at high dilu-
tion (1:100,000 or greater), some
decrease in signal intensity may be
observed. Antibodies may become
depleted in the small volume of
the ImmunoBlot channel. Increase the antibody concentration
2–5 fold.
Increase the antibody volume solution
per channel. To do this, insert standard
200 µl plastic pipette tips into both
entry ports for each channel. These will
serve as reservoirs for sample volumes
larger than the volume of the channel
itself, while incubating on the rocker.
Inject the antibody sample using a tip
that then remains in the entry port and
becomes such a reservoir.
Perform secondary antibody incubations
in a tray. (See Appendix A, Note 6.)
Troubleshooting
problem possible causes recommendations