Trouble Shooting your Western Blots

Here are some common problems that can occur with Western blots and how to troubleshoot them:

• No bands: This can be caused by a number of factors, including:
  • Not enough protein loaded on the gel.
  • The protein of interest is not present in the sample.
  • The antibody is not specific for the protein of interest.
  • The antibody has expired.
  • The antibody was not diluted correctly.
  • The membrane was not blocked properly.
  • The primary antibody was not incubated for long enough.
  • The secondary antibody was not incubated for long enough.
  • The substrate was not used correctly.
• Weak bands: This can be caused by a number of factors, including:
  • Not enough protein loaded on the gel.
  • The protein of interest is present in the sample, but at a low concentration.
  • The antibody is not specific for the protein of interest.
  • The antibody has expired.
  • The antibody was not diluted correctly.
  • The membrane was not blocked properly.
  • The primary antibody was not incubated for long enough.
  • The secondary antibody was not incubated for long enough.
  • The substrate was not used correctly.
• High background: This can be caused by a number of factors, including:
  • The membrane was not blocked properly.
  • The primary antibody was not diluted correctly.
  • The secondary antibody was not diluted correctly.
  • The substrate was not used correctly.
• Blurred bands: This can be caused by a number of factors, including:
  • The membrane was not washed properly.
  • The primary antibody was not incubated at the correct temperature.
  • The secondary antibody was not incubated at the correct temperature.
  • The substrate was not used correctly.

If you are having problems with your Western blots, try troubleshooting the problem using the tips above that we suggest. If you are still having problems email(info@precisionbiosystems.com)us or give us a call(888-490-4443).