Hi there,

I am currently panning against GFP and pre-depleting with PBS+0.5%BSA using the steps attached (modified from Barbas's protocol). GFP and BSA are immobilised on high-binding microtiter plates (corning: #3590) and I use different blocking buffers in all panning rounds.

I have conducted 5 panning rounds and wanted to check for specificity to GFP. Panning the amplified phage from round #4 and #5 against GFP, BSA (negative control) and PBS (negative control) showed approximately equal amounts of clones. Therefore, I guess the false positive rate/unspecific binding rate is high. Is there any obvious flaws in my protocol or is there another reasoning for this? Should i pick the clones for ELISA testing anyways?

For blocking the plates I use 0.5%BSA(9048-46-8) in PBS, 0.25%FISH skin gel in PBS, or Licor intercept blocking buffer. I also checked for non-infected bacterial growth and this is not case - I see no clones if the bacteria are not infected with eluted/ amplified phages.

I hope you can help.

All the best, Emil