Western Blot Doctor™ — Signal Saturation 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 signal saturation.

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.

A blot with ghost bands – Western Blot Doctor - Signal Saturation Issues

Ghost protein bands

A blot with signal saturation – Western Blot Doctor - Signal Saturation Issues

Target signal is too strong

 

 

Problem: Ghost protein bands

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A blot with ghost bands – Western Blot Doctor - Signal Saturation Issues

Likely Causes and Recommended Solutions Sample overloading
  • Decrease the total protein loading for each sample
Too much antibody
  • Decrease the concentrations of the primary and/or secondary antibodies
Rapid consumption of substrate
  • Switch to a less sensitive chemiluminescent substrate
Tips If you observe ghost bands on your blots, there are several likely causes. Sample overloading can be addressed by reducing the amount of sample loaded on the gel; prequantification of sample solutions by spectrophotometry or Bradford assay can be used along with dilution as needed to determine appropriate loading quantities. If the bands are due to excessive antibody binding, reducing the antibody concentration can help, as can switching to a different blocking agent; consult your antibody supplier for recommendations on appropriate blocking agents for your antibody. Ghost signals may also be due to the high sensitivity of the chemiluminescent substrate; check the manufacturer’s instructions for use and consider switching to a different substrate.
 

Problem: Target signal is too strong

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A blot with signal saturation – Western Blot Doctor - Signal Saturation Issues

Likely Cause and Recommended Solutions Sample overloading
  • Decrease the total protein loading for each sample
Too much antibody
  • Decrease the concentrations of the primary and/or secondary antibodies
Tips If you observe oversaturation in the target signal, the most likely cause is sample overloading; trials with smaller amounts of protein should be performed.

Oversaturation problems in general can be addressed by diluting the detection antibodies and/or reducing exposure time; several trials may be necessary to determine the optimum conditions for your samples.
 
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