Poor transfer quality:
The poor
transfer of proteins to the membrane can be visualized by staining the
gel after transfer
Possible causes:
1. Insufficient transfer time:
a. Large molecular weight proteins may require a longer transfer duration
2. Transfer current is too low
a. Large molecular weight proteins may be difficult to elute from the
gel at very low current settings
3. Improper buffer components
a. The conductivity of the transfer buffer is influenced by its components;
4. Excess methanol in the transfer buffer
a. Too much methanol in the transfer buffer decreases the transfer efficiency
of proteins from the gel to the membrane; however methanol aids in protein
binding to PVDF or nitrocellulose membranes – a balance is needed
(see protocols section)
5. Air bubbles between the gel and the
membrane
a. Air bubbles are not conductive and, therefore, interfere with protein
transfer – this can be seen as a ‘patchy’ transfer
with ‘holes’; to increase the surface contact between the
gel and the membrane, roll over the gel/membrane sandwich with a pipette
to drive out any air bubbles.
b. Inspect the sponge pads – if they are flattened, they may not
compress the gel and membrane together with enough force
6. PVDF membranes that have not been properly
hydrated
a. PVDF membranes are hydrophobic. In order for PVDF membranes to become
wet and to properly accept protein transfer, the membranes must be prewet
by using methanol
Detection Issues
High background;
low signal to noise ratio
Possible causes:
1.
Chemiluminescence – exposure too long
2. Insufficient blocking:
a. Increase the duration of non-fat dry milk application
b. Increase the concentration of NFDM used
c. Try blocking with normal serum of the host animal for the secondary
antibody
3. Insufficient washing
i. Increase the number or duration of wash steps to help remove non-specific
signal
ii. Use stronger detergents: if Tween-20 is used and background is high,
a stronger detergent such as NP-40 or SDS may provide a more stringent
wash
4. Antibody concentration too high:
i. If the primary or secondary antibody concentration is too high, perform
optimization experiments to determine proper dilutions