Optimization of blocking conditions for fluorescent Western blot

Blocking conditions are critical for reducing background and non-specific signals in Western blot (WB). For fluorescent WB, however, blockers may bring in additional autofluorescence, and optimal blocking conditions have been less well established. Here, the autofluorescence of Tween 20 is clarified, which is negligible when imaged wet, but could be significant when Tween 20 is dried on blots. In most situations, blocking with Tween 20 reduces background and/or improves specific signals. Systematical investigation of blocking conditions for fluorescent WB reveals that the autofluorescent background is determined by a combination of multiple factors including the blocker, the excitation/emission configuration, the membrane, and the H₂O (wet or dry), varying case by case. The commonly used protein or polymer blockers (milk, bovine serum albumin, fish gelatin, and polyvinylpyrrolidone) each have unique advantages and disadvantages concerning the autofluorescence, the blocking efficiency, and the cost. 0.005%–0.02% sodium dodecyl sulphate can be included to reduce non-specific bands and background caused by non-specific binding of antibodies, however, at the cost of also impairing specific signals. For poorly-performing antibodies, a pre-test might be necessary to identify a suitable blocker. This work provides a reference for cost-effective blocking conditions for fluorescent WB.