Western Blot Doctor™ — Background Issues

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Overview

The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to issues related to high or uneven background signal.

Other sections in the Western Blot Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.

 

 

Problem: High background across blot

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A blot with a high background – Western Blot Doctor - Background Issues

Likely Causes and Recommended Solutions Primary and/or secondary antibody concentrations are too high
  • Dilute the primary and/or secondary antibodies
Insufficient blocking of nonspecific sites
  • Increase the concentration of blocking reagent, e.g. 3–5% BSA, casein, skim milk
  • Decrease blocking time and/or temperature
  • Add 0.05% Tween-20 to blocking buffer
  • Prepare antibody dilutions in the same blocking buffer with 0.05% Tween®-20
Wrong blocking buffer was used; the primary and/or secondary antibodies bind to proteins in the blocking buffer
  • Test different blocking reagents (albumin, casein, skim milk, etc.) Do not use milk to block membranes when using an avidin-biotin system — milk contains biotin
Insufficient washing
  • Increase the number of washes and the volume of buffer used (min of 5 × 5 min)
  • Add 0.1% Tween®-20 to the wash buffer if it is not already included
Exposure time is too long
  • Reduce the exposure time
Membrane dried in the blotting procedure
  • Make sure membranes are wetted thoroughly
  • Ensure the membrane is adequately covered with liquid at all times to prevent it from drying
Contamination of reused buffers
  • Use freshly made buffers

Tips

If you observe high background across the blot, there are a number of likely causes. Careful attention to your handling and protocol steps is required, and multiple trials may be necessary to resolve this problem.

Recommendations: Start with 5% BSA and always include .1% Tween 20 in wash buffer, 1 hour blocking at room temp, agitate during wash steps.

The use of heat-sealed bags will prevent drying and contamination of the blot.

 


Problem: Blotchy background

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A blot with a blotchy background – Western Blot Doctor - Background Issues

Likely Causes and Recommended Solutions Part of the membrane dried out
  • Make sure membranes are wetted thoroughly
  • Ensure the membrane is adequately covered with liquid at all times to prevent it from drying
Insufficient access of washing buffer to certain blot area
  • Increase washing buffer volume
  • Avoid stacking too many blots in one incubation container
Insufficient access of washing buffer to certain blot area
  • Use clean or new transfer pads
Contamination on blot
  • Do not handle membrane with bare hands. Always wear clean gloves or use forceps
  • Make sure electrophoresis equipment, blotting equipment and incubation trays are clean and free of foreign contaminants

Tips

If you observe a blotchy background, the most likely cause is a dry membrane or insufficient washing; both problems are readily solved by strict adherence to the blotting protocol, ensuring that the blot is immersed at all times. Additional handling precautions should be taken to prevent accidental contamination from common laboratory sources.
 

Problem: Speckled background

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A blot with a speckled background – Western Blot Doctor - Background Issues

Likly Causes and Recommended Solutions Gel piece stuck on the membrane
  • Remove all residual acrylamide gel traces from the surface of the membrane
Clumps of the blocking reagent stuck to the membrane
  • Make sure that the blocking reagent is well dissolved prior to use
  • Add 0.1% Tween 20 to the blocking buffer
Antibody aggregation
  • Filter the antibody buffer through a 0.2 µm filter
  • Use a fresh aliquot of the antibody
  • Add 0.1% Tween 20 to the antibody buffer
Buffer contamination
  • Use fresh buffers
  • Filter buffers through a 0.2 µm filter before use
Tips If you observe a speckled background, the most likely causes are foreign material adhering to the membrane. Thoroughly remove all traces of the transferred gel and ensure that the blocking reagent is completely dissolved before blocking. Antibody aggregation can be prevented by microfiltration of the antibody solution prior to incubation.

Dirty scanner or cassette surfaces may also cause peckling. Cleaning all contact surfaces and wrapping blots in plastic wrap or “seal-a-meal” bags is a good safeguard against both contamination and blot dehydration.
 
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