I am looking to do phage display using a mammalian cell line as the immobilised target and was wondering how many cells I need to use. I have seen a whole range in the literature, but a number of these are using transfected cell lines to express their protein of interest. Where as I am aiming to identify a target from the panning experiments.
Hi, some times ago I found this article....I hope that It's right for your experiment.
But I have a question: do you have a specific target on the cell surface or you'll identify the target in a second time?
Thank you, this looks pretty useful. To answer your question no there is not a specific target on the cell
Do i understand it correctly, you going to pan a single protein or peptide fused to phage gIII.
How do you intend to identify/discriminate the target if you dont have +ve and negative cells???
if you only know the "bait" protein you could make a tagged molecule e.g biotin-peptide or protein, test whatever cell lines you have available using flow cytometry to see which has high surface expression of the unknown "prey" and then do pull-down followed by MS peptide fingerprinting.
Cross-linking using a reversible cross-linker like DSP might also help.
I will be using a random phage library which contains approximately 10^9 different clones containing random peptide sequences. Once panning rounds are completed I will sequence the remaining bound phage and try to identify a consensus sequence. Then use BLAST or a similar database to identify the proteins which were bound by the phage, if that makes sense? so I don't have a specific bait protein as such
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