I would like to coat E-cadherin (recombinant, His-tag) to a tissue culture treated 96-well plate (Cornings 3595: https://ecatalog.corning.com/life-sciences/b2c/US/en/Permeable-Supports/Inserts/Corning%C2%AE-96-well-Clear-Polystyrene-Microplates/p/3595?clear=true ). I try to verify the coating of plates by staining with an anti-E-cad antibody conjugated with fluorescence and observe the fluorescence. However, the trial is failed, and there are only dot-like fluorescence on the bottom of the coated well, with no difference from control one with PBS only.

I derive my E-cadherin protocol from a T cell activation protocol as shown here: http://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf. I tried to incubate coated wells with 50ul 10ug/ml E-cadherin solution in PBS or in sodium bicarbonate solution for 2h in RT or overnight in 4C fridge. Then I washed the wells with PBS once, blocked the wells with 5%FBS for 2h and stained the wells with antibodies ( https://www.biolegend.com/en-us/products/fitc-anti-mouse-cd326-ep-cam-antibody-4971, 500ug/ml, 1:100 in 1%FBS), which was incubated in 4C overnight. The stained wells were washed with PBS once.

Do you know anyway to verify the even coating of protein on a 96 well plate?

Furthermore, I check several protein-coating protocols for ELISA, which mention that tissue culture treated wells are not recommended for protein coating. But why in the verified T cell stimulation protocol mentioned above, the tissue culture treated 96-well plate is used?

I also find that it is mentioned that hydrophobic binding is the basis of passive adsorption for protein coating, and untreated polystyrene plates are used for protein coating, including in one protocol for E-cadherin coating: Article E-Cadherin-Coated Plates Maintain Pluripotent ES Cells witho...