Bio-Rad Trans-Blot® Cell - Manual (Page 21)

Immune-Specific Detection
Overall High Background
1. Blocking conditions are inappropriate.
• Match the blocker to the membrane. For example, nylon and PVDF
membranes require more extensive blocking, usually with non-fat dry
milk.
• Increase the concentration or blocking time as necessary.
• The blocker must be a pure protein. The blocker may be contaminated
with material that bind probes non-specifically.
2. Insufficient wash protocols are used.
• Increase the number, duration, or stringency of the washes. Include pro-
gressively stronger detergents in the washes, e.g. SDS is stronger than
NP-40 which is stronger than Tween-20. Also, include Tween-20
in the antibody dilution buffers to reduce nonspecific binding.
3. The blot is left in the substrate too long.
• Remove the blot from the substrate solution when the signal-to-noise
level is acceptable. Do not overdevelop. Stop the reaction immediately
by immersing the blot in dd H
2
O.
4. Contamination occurred during a previous step, e.g. electrophoresis
or transfer.
• Discard and remake the gel and transfer solutions.
• Replace or thoroughly clean contaminated fiber pads, if a tank blotter
was used. Excessive amounts of protein were loaded on the gel, or too
much SDS was used in the transfer buffer. Proteins can pass through the
membrane without binding and recirculate through a tank blotting sys-
tem. Reduce the amount of protein on the gel or SDS in the transfer
buffer. Add a second sheet of membrane to bind excess protein.
5. Primary or secondary antibody is too concentrated.
• Increase the dilution of the antibodies. Perform a dot-blot experiment to
optimize the working concentrations.
6. Incubation trays are contaminated.
• Clean the trays or use disposable trays.
Nonspecific Reactions between Bound Proteins and Probes
1. Primary or secondary antibody is contaminated with nonspecific or
species cross-reactive IgG.
• Use purified IgG first antibody fractions and affinity-purified blotting
grade secondary antibody.
2. Monoclonal antibodies may react non-specifically with SDS denatured proteins.
• Compare the binding of other monoclonal or polyclonal antibodies.
• Blot native proteins as a comparison.
3. Nonsense interactions are occurring due to ionic associations. For example,
avidin, a glycosylated protein, may bind to more acidic proteins on blots.
• Increase the ionic strength of the incubation buffers. Increase the num-
ber, duration or stringency of the washes. Include progressively stronger
detergents in the washes, e.g. SDS is stronger than NP-40 which is
stronger than Tween-20. Include Tween-20 in the antibody dilution
buffers to reduce nonspecific binding.
18