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Here are the seven most common errors, in order of frequency

After washing three times with TBST (Tris-buffered saline supplemented with 0.1% Tween-20), the samples were incubated for 1 hour at room temperature with a secondary antibody

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Also, if youre using a laminar flow hood or clean bench, sterilize the workspace with appropriate disinfectant (and UV light, if available, prior to work)