Western blots are complex and offer many opportunities for something to go wrong. Can you tell what went wrong with this blot?
What caused the large, dark smudge on the lower left-hand side? In this case, the source of the smudge is a fingerprint from a bare hand. Our fingers are covered in oils. When we touch something, we leave oils behind as a fingerprint. Fingerprints will fluoresce in the 488, 520, 700, and 800 nm channels, which means they will appear as a dark smudge when imaged on an Odyssey® Imager.
How do I avoid leaving fingerprints?
Unlike a burglar, simply wearing gloves will not solve this issue. While gloves prevent you from leaving your fingerprints behind, they can introduce other contaminants to your blot. Ideally, you want to minimize blot handling. When it is necessary to move the blot, only handle it by the edges using forceps cleaned with methanol.
Why do fingerprints matter?
If the fingerprint is nowhere near any bands or lanes to be quantified, it’s just an aesthetic issue and should not affect quantification. However, if the fingerprint is near or covering an area to be quantified, it will affect quantification. This means you will need 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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