I am a grad student new to working with antibodies. I want to know if anyone can help me determine how best to find the optimum concentration for my primary and secondary antibodies. Ive noticed that some people perform titrations...
I have antigen that is 0.5mg/ml
primary antibody that is 0.5mg/mL and my secondary antibody is 0.5mg/ml as well. It was suggested to me to have a 1:50 dilution of the primary and 1:500 dilution of the secondary.
In order to determine if this is the best dilution would i set up a series of titrations in a well tray?
Also, can you tell me why you would use more primary than secondary? Is this bc of the bindng constants?