Colony lift followed by probing with a peptide; does this exist? - (May/30/2006 )
Hi folks, this is my first post, so let me know if I'm not getting anything
I have a peptide that I would like to introduce random mutations into in the hopes of generating higher affinities. What I would like to do is use an error prone polymerase and pcr out the peptide gene and ligate it, followed by transforming into BL21 e.coli cells.
Here's the part I haven't seen any literature on yet. I want to transfer the entire transformation plate (presumably with all the different mutants) onto a membrane and probe with my known ligand, conjugated to biotin, which I will then detect with streptavidin-AP, hopefully finding the tightest binders lighting up the most. I've seen protocols for colony lifts, but subsequent steps always involve hybridization with nucleic acids. Can you probe lifted colonies with peptides?
Thanks for any help!!
1. your target should be on the outside of the cell
2. most probably your peptide might also stick to the call surface, so you'll probably select peptides binding cell surfaces
3. you have to make sure, you'll get the dna corresponding to your binders back.
yes it exists. It's called colony blotting.
you transfer the colonies on nitrocellulose membrane, transfer the membrane on agar plus IPTG if necessary, then transfer the membrane on several buffers to lyse the bacteria and then procede like a western-blot.
you will find the detailed protocol on qiagen website.
yeah, but in that case, you detect via antibody, which is a very specific interaction. i dunno if a similar approach will work on binding peptides
Is it worse to try? It's a one day procedure. Of course, you need to set up with non expressing bacteria, and the bacteria expressing the WT peptide.
no no no ... of course only the experiment is the prove. i would always try, if i have an idea which could work. only thing i was trying to point out is the fact, that he is trying to do evolution, if i understand correctly. and as far as the peptide will not bind so tight/specific as the AB, he'll probably will end up with a peptide against something completely different than he expected.
i remember someone who was selecting against a sugar from a library, and ended up having selected binders against the column material the sugar was on
Thanks for your quick and helpful replies. I think I now have enough info to put together a pilot experiment. Of course, if anyone thinks of anything else, I'm open to more!