Protocols, optimization tips,
and more for Western Blot.
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Western Blot Troubleshooting: Background Issues
The background of a western blot does not always appear clean and flawless. Blotches, streaks, and spots are all common artifacts that can make it hard to interpret and publish your results. These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer. Follow these tips to identify and solve the cause of your imperfect western blot background:
|Blotched background||Uneven antibody distribution||Agitate during incubation to coat the membrane evenly in incubation buffer|
|Membrane dried out unevenly||Make sure membrane is thoroughly wetted before starting the protocol|
|Ensure the membrane does not dry out during any step|
|Uneven wash/incubation buffer coverage||Increase volume of wash and incubation buffers|
|Do not stack membranes during incubation|
|Flecked background||Secondary antibody aggregation||Increase secondary antibody dilution to prevent aggregation|
|Spin down or filter out antibody aggregates|
|Clumps of blocking buffer binding secondary antibody||Use fresh blocking buffer|
|Increase Tween 20 concentration of blocking buffer|
|Filter blocking buffer before use|
|Use a different blocking reagent, such as albumin, BSA, or casein|
|Wash membrane with wash buffer before antibody incubation|
|Buffer contamination||Mix new buffers|
|Filter buffers before use|
|White spots with no protein transfer||Air bubbles trapped between gel and membrane during transfer||Carefully squeeze out bubbles from between membrane and gel using a sterile glass rod|
|Use enough buffer to saturate the membrane|
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