Hi everyone!!! The output of the succesives round don't increase. Do you know what could happen?
many replies possible:
- library intrinsically unable to bind your antigen
- too little antigen used
- too harsh washing steps
- stickiness of phage particles that bind tube surface with aspecific binding overweighing antigen-specific binding
- ... many more
try panning on biotinylated antigen with elution by reduction (antigen biotinylated with a reducible biotinylating agent). usually elution is much more specific. try performing panning in glass tubes to decrease phage aspecific retention (I never tried this yet)
phage display can be very tricky. Before starting you should make sure that the library stock you are using is fresh and that it well represents the theoretical size of the library (n° of different clones). good luck
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