What might be the problem if the bands are not appearing in Western Blot?
1 Answer
Many...
Explanation:
There are many reasons why bands may not appear on a western blot. Have the sample and antibody combinations worked in the past?
Below are just some that I can think of at the moment that may cause bands not to appear:
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Did the protein transfer from the gel? Try staining the membrane with something like ponceau S or amido black to see if the bands are present. Sometimes, you can see the protein bands on the membrane by wetting it and holding it at an angle to the light.
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Is the primary antibody working? This is a tough one to test and the only way you can is by including a positive control where you know you have the protein of interest present. If you have some of the protein of interest you could try spotting it onto the Western blotting membrane (i.e. you don't run the gel) and seeing if you get a result if you process the membrane as if it were a western blot.
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Is the secondary antibody recognising the primary antibody? Again, a tough one to test. About the only test you can do is the spot test mentioned above in 2.
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Is the "detection system" working? Depending upon the detection method you are using you could try spiking in some of the secondary antibody to see if the detection solution, and also the triggering agent/enzyme on the secondary antibody, is working. That is, can you trigger the reaction just with the secondary antibody?
Finally, it could be as simple as one of the solutions used during the probing of the plot being made up incorrectly. For example, if the salt concentration is wrong in the buffer then this may cause the antibodies to be released from the blot. The same would also happen if the pH of the buffers were incorrect.
The "weirdest" cause for a western blot not working that I have personally experienced was when we changed the supplier of the milk powder we used to block the membrane. The powder from the new supplier contained a phosphotyrosine phosphatase which removed all the phosphate groups that we were trying to detect with our anti-phosphotyrosine antibody.
