6. Methanol in the transfer buffer is restricting elution of proteins from the gel. Elimination
of methanol results in increased transfer efficiency, but it also diminishes binding to
nitrocellulose. Use PVDF.
7. Protein is precipitating in the gel. Try using SDS in the transfer buffer. SDS can increase
transfer efficiency, but can also reduce binding efficiency to nitrocellulose and affect
reactivity of some proteins with antibodies.
B. Swirls or missing patterns on blot; diffuse transfers
1. Contact between blot membrane and gel is poor. Air bubbles or excess moisture remain
between the blot and gel. Use a test tube or pipet to roll over the membrane carefully in both
directions until excess moisture and air bubbles are removed from between gel and membrane
and complete contact is established. Use thicker filter paper in the gel/membrane sandwich.
Make sure that there are no air bubbles trapped between the filter paper and the gel.
2. The gel is not completely equilibrated in transfer buffer. Gel must be properly washed in
transfer buffer to avoid shrinking or swelling during transfer. Increase time or number of
washes.
3. If multiple gels are being transferred simultaneously, cross-contamination may be
occurring. Use a smaller size pore dialysis membrane to separate gel/membrane
sandwiches. Use PVDF to more completely bind small pieces.
4. Power conditions are too high. Reduce the voltage. Check the buffer conductivity; improp-
erly prepared buffer will result in excessive power delivered to the cell.
8.2 Poor Binding to Nitrocellulose Membrane
1. Proteins separated by SDS-PAGE require 20% methanol in the transfer buffer for
optimal protein binding. Make sure the buffer contains the proper amount of methanol.
2. Proteins may be transferring through the nitrocellulose, driven by the high field strength
of the plate electrodes. Use Zeta-Probe membrane (higher binding capacity) or 0.2 micron
nitrocellulose (smaller pore size). Transfer using the Trans-Blot cell or the Mini Trans-Blot
cell with standard platinum wire electrodes.
3. Protein >15,000 daltons may show diminished binding to 0.45 micron nitrocellulose, or
may be washed from the membrane during assays. Use Zeta-Probe membrane or
0.2 micron nitrocellulose. To increase stability of binding, proteins can be cross-linked to
nitrocellulose with glutaraldehyde.
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4. Proteins can be removed from nitrocellulose by SDS, NP-40, and several other
detergents. Use Tween-20 detergent in wash and antibody incubation steps. Reduce or
eliminate detergents from buffers. Try glutaraldehyde fixation.
5. SDS in the transfer buffer will reduce binding efficiency of proteins. Use 20% methanol
in the transfer buffer and equilibrate the gel in methanol buffer prior to transfer.
8.3 High Background After Incubation with Antibody Probes;
Nonspecific or Nonquantitative Detection
For a complete troubleshooting guide to Immun-Blot assays, consult the Immun-Blot
assay kit manual or the Zeta-Probe instruction manual. If using other detection kit, consult
manual or contact manufacturer.
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