Bacterial Contamination in Wash or Incubation Buffers (ELISA (Signal Problems))

Question

Accepted Answer

Sporadic signal loss or high background that worsens over time during multi-day experiments. Cloudy or turbid buffer appearance. Inconsistent results when using same buffer batch. Common causes: 1) Bacterial growth in buffers stored at room temperature or 4°C without preservatives 2) Contamination introduced through non-sterile pipette tips or reagent bottles 3) Bacterial proteases degrade antibodies and protein targets in buffer 4) Bacterial components bind non-specifically to plate and increase background Solutions: 1) Use fresh, sterile PBS or manufacturer-supplied sterile buffers for each experiment 2) Prepare wash and incubation buffers fresh on day of use; discard any leftovers 3) Add preservatives to buffers if stored: 0.02% sodium azide (avoid with HRP) or 0.05% ProClin-300 4) Use sterile technique: filter buffers through 0.22 µm membrane and use sterile pipette tips 5) Store prepared buffers at 4°C for maximum 1 week; inspect for cloudiness before use