On day 6 (final day) of the phage display procedure, I allowed the bound phage to elute with gentle rocking using glycine-HCl (pH 2.2) for 35 minutes instead of the recommended 10-20 minutes and then neutralized it with Tris-HCl (pH 9.1). Will that have an adverse effect on my phages and affect the following phage titering/plaque counting experiment?
Hi Omar, you will find out by phage titration how severe your new conditions were. The only thing you need to be afraid of is that the eluted phages are not infectious and thus you may loose good binders and possibly diversity. In most cases you don't need such harsh conditions as most peptide binders are rather weak (except for some peptides binding to antibodies in case you are doing epitope identification). The good thing about phage display is that it is fast and that you get a lot of information from the titers of the eluted phage to guide your experiments. Good luck!
Thank you Markus! If it can possibly affect the infectability of the phages, then I’d rather err on the side of caution and repeat day 6, as the next step will be DNA sequencing which is preceded by phage amplification in E.coli.
Ps: I was still able to detect around 30 plaques the day after these severe elution conditions upon a 1000 fold dilution, however, it’s not conclusive evidence to preclude the possibility of having lost potentially good binders.
Omar, if you have the time you may want to compare the "harsh" vs. "soft" eluted clones. How does the total number of eluted phages in round 6 compare to those eluted in the previous rounds? I also suggest that after grouping the clones according to their sequences you select a number of the to amplify, titrate them and compare them in phage ELISA. It is quite easy to select peptides that bind to your target while on the phage particle, however it can be rather challenging to also get candidates that bind well as free peptide or fusion protein. Good luck!
The harsh eluted clones yielded 28 plaques, which is almost the same amount of plaques yielded in day 4 (27) where 1000 fold dilution was applied as well. Also, I repeated day 6 again applying soft elution conditions this time i.e. 15 min glycine-HCl buffer elution, however, I obtained significantly more than 100 plaques bearing in mind I applied the same dilution rate (1000 folds). Therefore, I plan on repeating the phage titration experiment using 10^3-10^5 dilution rates. Following the DNA sequencing procedure, I plan to synthesize the peptides and test their binding affinities against my target using ITC instead of ELISA.
(My target was biotinylated and panned against streptavidin coated plates, which might help eliminate the possibility of the phage peptides binding onto the plastic surface of the plates.)
Hi Omar, when you get the results from the sequencing check the literature for described binders to streptavidin. You can also do a competition phage ELISA to see if you can inhibit the binding of the phage with soluble target. This would give you additional data for deciding which peptides to select for further experiments. Good luck!
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