Western Blot Troubleshooting Background Background Blotchy, Flecked, Or Dirty

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:

Problem Cause Solution
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