*3.2.2. Protein binding*

Protein binding to dispensing components is an important point to consider in the imple‐ mentation of biochemical assays, particularly at low protein concentrations. In some instan‐ ces, enzymes appear to be inactivated over time when dispensing multiple plates using a liquid handler, when in reality the enzymes have been depleted from the solution due to non-specific binding to plastic, silicone and other polymer-based surfaces. This effect is am‐ plified when dispensing sizeable number of plates, as there is larger exposure time of the assay components to the surfaces of reagent reservoirs and dispensing cassette elements. In order to circumvent this problem, blocking reagents can be added to the buffer, plastic sur‐ faces can be coated, or a combination of both. The two major types of blocking reagents are detergents and proteins. It is preferable to use non-ionic detergents such as Tween-20, Triton X-100 or Nonidet-P40. Among the most widely-used protein blockers are bovine serum al‐ bumin (BSA) and casein. Protein blockers are better suited for coating surfaces, as detergents can be easily washed away. Typical working concentrations for detergents range from 0.01 to 0.1%, while protein blockers are used between 0.1 to 3 %. The selection of the appropriate type of blocking reagent and concentration is central to a robust assay. Other less common blocking reagents include polyethylene glycol (PEG), polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP). Additionally, the use of glass reagent reservoirs is recommended.
