26
- nitrocellulose
•
Ensure methanol concentration does not exceed 20%
(v/v).
Proteins may be transferring through nitrocellulose.
•
Use PVDF or smaller pore size (0.2µm) nitrocellulose.
•
Overlay an extra piece of nitrocellulose over membrane to
determine if proteins are migrating through the membrane
directly in contact with the gel.
Proteins <15kDa have reduced binding to 0.45µm
nitrocellulose or may be washed from membrane during
assays.
•
Use PVDF or nylon membrane, which have higher binding
capacities.
•
Use Tween-20 detergent in the wash and antibody
incubation steps. Reduce or eliminate the more stringent
washing steps.
SDS in transfer buffer reducing binding efficiency
•
Reduce or eliminate SDS concentration
Membrane incompletely wet
•
White spots indicate dry areas where protein will not bind.
•
If soaking does not occur immediately following immersion
in transfer buffer, heat distilled water to just below boiling
point and soak membrane until entirely wet.
Poor binding to membrane
PVDF
Membrane is not completely wet
•
Because of hydrophobicity of PVDF, the membrane must
be soaked entirely in methanol before equilibration in
aqueous buffer
Proteins might be transferring through the membrane
•
Decrease voltage if transferring under high intensity
conditions
•
Overlay an extra piece of PVDF over membrane to
determine if proteins are migrating through the membrane
directly in contact with the gel.
Membrane might have dried during handling
•
Fully wet membranes have a grey translucent
appearance. White spots will form on the surface if the
membrane has been allowed to dry. As proteins will not
bind to dry spots, re-soak the membrane in methanol and
re-equilibrate in transfer buffer
SDS in transfer buffer reducing binding efficiency
•
Reduce or eliminate SDS concentration
Power is too high
Always check current at the start of the run, for the current
might be too high for a given voltage setting. Improper buffer
preparation can also result in high conductivity and excessive
power generation. The current setting should not be allowed
to exceed 2000mA.
Immune-specific detection
Overall high background
•
Reduce antibody / protein sample concentration
