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No Bands Observed in western blot, what should I do?

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2 Answers

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Too many possibilities to make a suggestion.

  1. Is there protein on your blot? Maybe you transferred in the wrong direction.
  2. Did you add secondary antibody?
  3. Is you primary antibody still functioning?
  4. Did you expose long enough?
  5. Did you block with too much milk/BSA/whatever? Low abundance proteins can be covered.
  6. Did you load enough protein?
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western blot is evil, isn't it? – glucose♦ Feb 2 at 18:35
A little less evil than cloning, in my experience ;) – Kyle Legate Feb 3 at 9:17
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Insufficient antibody

Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration). Antibody may have lost activity. Perform a Dot Blot.

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