Tween-20 Concentration Outside Optimal Range (Western Blot (CST Guide))

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Signal intensity is lower than expected or background is elevated. Changing Tween-20 concentration in wash/incubation buffers significantly affects results. Common causes: 1) Tween-20 concentration higher than 0.1% in 1X TBS wash and incubation buffers compromises antibody binding 2) Tween-20 concentration lower than 0.1% insufficient to reduce non-specific binding and background 3) Use of PBS instead of TBS weakens signal intensity with some CST antibodies Solutions: 1) Maintain Tween-20 at exactly 0.1% in 1X TBS for all blocking, antibody incubation, and washing steps 2) Use 1X TBS rather than PBS as base buffer; PBS may weaken signal with some antibodies 3) Prepare fresh buffer with accurate Tween-20 measurement: 0.1% = 1 ml Tween-20 per 1 liter TBS 4) Verify buffer pH and composition match CST recommendations: 1X TBS/0.1% Tween-20