Precipitate Formation in Wells Upon Substrate Addition (ELISA (High Background))

Question

Accepted Answer

Visible precipitate or cloudiness appears in wells immediately or shortly after substrate addition. Absorbance readings may be abnormally high or variable. Common causes: 1) Substrate concentration too high causing precipitation 2) Sample concentration too high leading to protein aggregation 3) Incompatibility between substrate components and sample matrix 4) pH or ionic strength mismatch causing precipitation Solutions: 1) Decrease substrate working concentration 2) Dilute samples further to reduce protein concentration 3) Centrifuge samples before use to remove particulates 4) Filter substrate solution through 0.22 µm filter before use