Unusual gel or band apperance in western blot
Explore potential causes and solutions for unusual gel or band appearance in western blot.
Why are there black dots or speckled background?
The blocking reagent has clumped together, and antibodies are binding to it.
This binding will appear as dots of positive signal.
- Filter the blocking agent.
The gel or reagents are contaminated with bacteria.
Like buffers, reagents can also be easily contaminated in the lab by a variety of sources, including bacterial sprores in the air or stray fibres in vials.
- Make sure you use fresh, sterile buffer (eg our sterile PBS).
Why are there white spots or smudges?
Air bubbles were trapped against the membrane during transfer.
Bubbles will appear as uneven white spots.
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Make sure you remove any air bubbles caught between the gel and the membrane during transfer.
You can do this by lightly pressing down on the stack with a small roller.
You should be able to see any bubbles after checking the success of the transfer with Ponceau S.
Why do bands appear white (if using ECL detection)?
Primary and secondary antibody concentration may be too high.
If the antibody concentration is very high, then the substrate is consumed very quickly. This means very little light is absorbed at this point, leading to a white band when you image the blot.
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Make sure you remove any air bubbles caught between the gel and the membrane during transfer.
You can do this by lightly pressing down on the stack with a small roller.
You should be able to see any bubbles after checking the success of the transfer with Ponceau S.