Coating your own ELISA plate

Coating overnight at 2 to 8 °C

The common pattern is to dilute the capture antigen or antibody in coating buffer, add a fixed volume to each well, seal the plate, and leave it overnight at 2 to 8 °C. The long, cold incubation lets protein adsorb slowly and evenly rather than drying at the edges. Keep the volume consistent across wells, seal against evaporation, and do not stack plates in the cold room in a way that leaves some plates colder than others. Bring the plate back to room temperature before the next step.

Coating buffer basics

Carbonate buffer at pH 9.6 is the common choice for coating. The alkaline pH favors adsorption of most proteins onto polystyrene, which is why it became the default. Phosphate buffered saline is also used, and some proteins coat better in one than the other, so the buffer is an optimization variable, not a fixed rule. Whatever you choose, make it fresh or verify the pH, use the same buffer across a study, and record it, because a silent buffer change can look exactly like a reagent problem.

How much capture reagent

Coating concentrations are assay specific. Published methods commonly fall in the range of 1 to 10 micrograms per milliliter for the capture reagent, but the right value for your molecule must be found by titration, coating a plate across a dilution series and reading the signal and background. More capture protein is not automatically better: past saturation it adds cost and can raise background. Optimize for the best signal to background ratio, not the strongest raw signal.

Blocking: covering what the capture layer missed

After coating and washing, some of the well surface is still bare plastic, and any protein added later, including your detection antibody, will stick to it and read as background. Blocking fills those sites with an inert protein solution before the sample goes in. Common blockers include bovine serum albumin and casein based solutions, left on for an hour or more. The blocker has to be compatible with your assay; a blocker that contains the analyte, or a protein your antibodies cross react with, creates the background it was meant to prevent.

Drying and storing coated plates

If you coat plates in batches, dry them after blocking and store them sealed with desiccant at 2 to 8 °C. A stabilizer solution before drying helps the capture layer survive storage. How long a stored plate stays good is assay specific and must be validated with controls run on fresh and stored plates; do not assume a stored plate performs like a fresh one. Label every stored plate with the coating date, reagent, and concentration, because an unlabeled plate found in the cold room is a rerun waiting to happen.

Related guides

• the 384 well plate guide: formats, a 384 vs 96 comparison, and templates
• the 96 well plate guide: types, volumes, and a free plate map template
• best deep well plates by job, plus sealing and KingFisher fit guides
• the cell culture plate guide: treated vs untreated, formats, and surfaces

Common questions