Observation
Possible cause
Recommended action
Protein not
binding/transferring to
membrane (PVDF).
PVDF membrane was
dry/partially dry.
Regions where PVDF membranes are dry
appear whiter than places where the
membrane is wet. Remove the membrane,
reactivate in 100% methanol, and rinse in
water before reapplying to the transfer stack.
High background.
TBST buffers were used for
washing.
Use PBST or WesternBreeze
™
wash solutions.
Signal intensity is similar for
different protein loads after
detection.
High protein load (detection of
is not within the linear range).
Since the immunodetection sensitivity is higher
for dry blotting with the iBlot
™
2 Gel Transfer
Device than for semi-dry or wet blotting, we
recommend that you decrease the protein load,
use more diluted antibody, or perform
detection for shorter time. You may need to
perform some optimization based on your
initial results.
Corrosion of the Top Stack.
Incorrect placement of the Top
Stack.
Be sure the Top Stack is placed correctly with
the copper electrode facing up. Avoid placing
the Top Stack in the inverted position.
Membrane and the gel turns
blue.
Longer transfer times resulted
in the deposition of copper ions.
Be sure to perform the transfer for the
recommended time for each gel type.
Green discoloration of
membrane edges.
Copper ions carried with liquids
reached the membrane.
These deposits do not interfere with
downstream processes. The stained regions
can be cut away, but membrane washing
typically results in their removal.
Bottom Stack transfer gel
melts to a viscous blue
solution.
Membrane was trimmed to fit
the gel size, resulting in direct
contact between the Top and
Bottom Stacks.
Always maintain the membrane size identical
to the transfer stack. Transfer quality is not
affected by smaller gel size compared to the
membrane.
Appendix A
Troubleshooting
Introduction
A
iBlot
™
2 Dry Blotting System User Guide
47
