What is the most effective approach to minimize edge effect in a 96well elisa? All reagents & plates are brought to room temperature before using. Plates are covered during incubations, stacking no more than 4 high. Coating takes place at 37C O/N in a humidified container. All other incubations take place at RT (all 1 hour incs.), but not in a humidified container, just on the benchtop with lids on the plates. Room temperature is consistant. Substrate is diethanolamine, not incubated in the dark. Plates are medium bind Greiner. Edge effect happening is a larger than expected drop from the top row to the second row in a 2-fold serial dilution down the plate, ends up more like 2.5-3 fold, other dilutions below the 2nd well come down as expected compared to the well above it.
1- Can coating at a shorter incubation(5 hours) period at 37 help or keep O/N inc with 4C instead? ODs may not be high enough, this is a lower binding assay to begin with, many time only getting 1 or 2 usable ODs for a sample-which makes it unreliable to quantitate when the edge effect happens.
2- Can using a high bind plate help, so that incubations might be shorter?
3- Can using a rotator for the plates during incubations help?