I coat 1 ug/mL of the Her2 protein with 100 uL 100mM carbonate buffer to my flat bottomed well and have only had a positive result once out of 8 times. I have tried coating at 4 deg C overnight, 37 deg C for 1 hour, shaking for 1 hour at RT and with no avail. When I baked my plate at 50 deg C I was able to get a strong signal but I believe that the protein was no longer in the native state. I know my ELISA works but for some reason it will not coat. Please help, thank you.
Hi there,
I guess what you call "amine plate" is an amine functionalized plate, right? This product is actually made to react covalently with carboxylate in presence of carbodiimide. If you are doing this, the reaction is favoured at acidic pH (and possible at neutral pH) but impossible at basic pH. What is surprising is that you use a carbonate buffer which is basic... If you want to do this kind of crosslinking, use a near neutrality buffer for a better yield.
The carbonate buffer is usually used for coating PVC plate (non covalent binding), so you might be making a mistake because mixing protocols.
Elaborate on your system, please. What is the amino plate? Are you looking for covalent or electrostratic adsorption?
The plate is a DNA-BIND Amine binding 96 well plate. When I coated a GFP protein to the plate it had a strong signal compared to my Her2 protein. That indicates to me the the protein was able to be covalently linked to the well. Would the conformation of the protein somehow affect the binding?
A strong signal does not represent covalent binding. Anyhow, the chemistry of union must be known. As stated above i dont think we are using carbodiimides. It could be that the plate is either activated with nhs or aldehyde groups or something else. You must know what it is such that your conditions favor the reaction.
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