Troubleshooting Antibody Non-specific Binding using Albumin as a Blocking Reagent
Posted by Joseph Abdalla


There are many kinds of problems that are common in western blotting. These problems could lead to unexpected western blot results, which could be unreliable and require troubleshooting.  Two of the main problems that are commonly encountered in Western Blotting are High Background and Non-specific binding showing many bands.

Non-Specific Binding:  is experienced when you see not only the protein band in question becoming visible but multiple unexpected bands becoming visible as well.  This is generally caused by having antibody concentrations too high or insufficient blocking. 


The simplest fixes for the majority of cases are:

  1. 1.  Reduce the 1o and 2o antibody concentrations, especially when using enhanced chemo luminescence.
  2. 2.  Reduce the amount of total protein loaded into the gel, DO NOT OVERLOAD THE GEL
  3. 3.  Increase the concentrations of the blocking agent by 2-5%
  4. 4.  Increase the blocking time.
  5. 5.  Increase the blocking temperature.
  6. 6.  Add 0.05% - 0.1% Tween 20 to blocking buffer.
  7. 7.  Increase the NaCl concentration in the blotting buffer.
  8. 8.  Use the blocking buffer to make your antibody dilutions.
  9. 9.  Avoid SDS in the assay.
  10. 10.  Addition of a protease inhibitor to samples made from homogenized tissues.
  11. 11.  Minimize Freeze/Thaws of your sample.
  12. 12.  Try different 1o and 2o antibodies (A control of just the 2o alone without the 1o)
  13. 13.  Check the reagents for bacterial contamination.


High Background:  Is seen as a stain across the whole membrane where the antibodies have bound to your blocking reagent.  This is generally caused during blocking and can be remedied by:

  1. 1.  Increase blocking time and temperature.
  2. 2.  Increase the Tween 20 to  0.1% - 0.5% in the blocking buffer.
  3. 3.  Add 0.15-0.5 M NaCl to the wash, blocking, and blotting buffers.
  4. 4.  Avoid Non-fat dry milk, substitute with 3% BSA.
  5. 5.  Increase the number, temperature, and length of the washes.
  6. 6.  Try different 1o and 2o antibodies that are different from the sample species.
  7. 7.  Lower the reaction and developing times.
  8. 8.  Reduce the 1o and 2o antibody concentrations.

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