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Western Blotting Troubleshooting

Problem: Poor transfer quality: The poor transfer of proteins to the membrane can be visualized by staining the gel after transfer.
             Possible Causes: 
 Insufficient transfer time:
a. Large molecular weight proteins may require a longer transfer duration

Transfer current is too low
a. Large molecular weight proteins may be difficult to elute from the gel at very low current settings

 Improper buffer components
a. The conductivity of the transfer buffer is influenced by its components;

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;

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

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.

            Problem: High background; low signal to noise ratio
P          Possible Causes: 
Chemiluminescence – exposure too long
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

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
Antibody concentration too high:
i. If the primary or secondary antibody concentration is too high, perform optimization experiments to determine proper dilutions