Western blots are complex and offer many opportunities for something to go wrong. Can you tell what went wrong with this blot?
Both A and B are air bubbles but with different causes. Bubble A is the result of air trapped between the gel and the membrane during transfer. Bubble B is the result of air trapped between the membrane and the scan surface during imaging.
How can I avoid bubbles?
To ensure you have removed air bubbles before the transfer process, carefully roll out your transfer sandwich using a roller, glass tube, or similar device. When imaging your blot on a glass scan surface, such as on the Odyssey® M Imager, roll out any air bubbles and cover the blot with a silicone mat to keep it flat.
Why do bubbles matter?
Bubbles cause image distortion. Depending on the type of bubble and where it occurs, quantification may not be possible, and you may need to redo the blot. Bubbles that are not near any lanes or bands to be quantified, such as B, are only an aesthetic issue and should not impact quantification.
You will need to address bubbles near an area to be quantified. A bubble between the scan surface and membrane only requires reimaging the blot. However, a bubble that occurred during transfer will require you to redo the blot.
For more troubleshooting examples and Western blotting tips, check out Good Westerns Gone Bad: Tips to Make Your Fluorescent Western Great.
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