16
Appendix
Troubleshooting
Problem
Possible Cause
Solution
No power (the digital
display remains black
when the power is
turned on)
AC power cord is not connected.
Fuse has blown.
Check AC power cord connections at both ends.
Use the correct cords. Replace the fuse
If the problem still persists after verifying that
correct power cord is used and the fuse is
replaced, contact Technical support.
Buffers leak from the
trays and tank
immediately
Valves remain open.
Turn instrument off, wait at least 5 sec and
turned instrument on. After initialization valves
will be closed.
Weak or no signal from
the blot
Detection step missed or
detection reagents not working.
After the blot processing is complete, perform
the detection step using your standard detection
reagents and protocol manually. Make sure the
detection reagents are functional.
Insufficient incubation with
detection reagent
Remove blot from detection reagent when
signal-to-noise ratio is acceptable.
Poor or incomplete transfer
Make sure transfer apparatus and membrane
sandwiches are assembled correctly. Use
appropriate transfer times. After blotting, stain
membrane to measure transfer efficiency.
Protein of interest ran off the gel
Use positive control and/or molecular weight
marker to match gel separation range to size of
protein being blotted. After blotting, stain
membrane to measure transfer efficiency.
Incorrect reagents added or
incorrect containers are filled
Make sure that primary and secondary antibody
are added to correct containers and number on
antibody container in the tank and tray match
each other.
Sample too dilute
Load the larger amount of protein onto the gel
or increase concentration of proteins.
Poor retention of proteins or
protein weakly bound to
membrane
Use membranes with appropriate binding
capacity. Dry PVDF membrane after protein
transfer to ensure strong binding of the proteins.
Inactive or overly dilute primary
or secondary antibody
Determine antibody activity by performing a
serial dilution using six trays or dot blot.
Increase antibody concentration as necessary.
High background on the
blot
Film overexposed or became wet
during exposure
Decrease exposure time or allow signal to
further decay. Prevent leakage of solutions by
encasing membrane in transparency film and
blotting excess substrate from edges before
exposure.