17
Short blocking time or washing
intensity
Increase blocking time and the number of
washes
High concentration of primary
and/or secondary antibody
Determine optimal antibody concentration by
performing dilution series using all six trays.
Decrease antibody concentration as necessary.
Protein is overloaded
Reduce load or dilute concentration of sample.
Membrane, solutions, trays, or
antibody containers are
contaminated
Use clean glassware and purified water to
prepare solutions. Wear clean gloves at all
times. Use forceps when handling membranes.
Run cleaning protocol with cleaning buffer,
increase the concentration of cleaning buffer
two times
Protein is overloaded
Reduce load or dilute concentration of sample.
Non-specific
binding too high
Insufficient removal of SDS or
weakly bound proteins from
membrane after blotting
Follow proper protocol for membrane
preparation before immunodetection.
Short blocking time
Increase blocking time.
Affinity of the primary antibody
for the protein standards
Check with protein standard manufacturer for
homologies with primary antibody.