Hi Chirantan Debnath . Just looking at the gels of both products gives me pause about the homogeneity of their CD63 prep.

But, if you are only interested in antibody recognition of immobilized CD63 in ELISA, I would recommend that you coat at your normal concentration (1 ng/ul) and let it dry out (or put it in a 37oC oven to dry out). The drying will force whatever protein is there to stick to the plastic ELISA plates.

Hi, Chirantan Debnath.

Try drying to strengthen the binding of proteins to the bottom of the plate.

Hello Chirantan Debnath

You may try changing the ELISA plate. Also, incubate the plate overnight in coating buffer for not more than 16 hours at 4 degree C. Which coating buffer are you using? Try using bicarbonate buffer pH less than 9.0. If 1ng/ul works fine with you continue using the same concentration. Otherwise, increasing the concentration would also help.

Good Luck.

Dear Chirantan, I'd advise against direct coating of microplates, unless you're satisfied with qualitative data. The surface of microplates is far from homogenous and you'll observe high well to well variability. Plus, a lot of your antigen molecules will stick flat to the surface and become inaccessible.

Better coat the plates with an antibody for a sandwich ELISA or even coat the plates with an anti species antibody against the first antibody of the sandwich.

Another options is biotinylating the catching protein and to coat the plate with streptavidin.