In our lab we perform routinely biopanning experiments with random phage display libraries, but we have never done negative selection.... The time has come I believe, and I wonder should if I should do it before or after positive selection? does it matter?
Another issue bugging me is should I do it starting from the first round? I saw in NEB protocol (https://www.neb.com/protocols/1/01/01/phage-display-solution-phase-panning-with-affinity-bead-capture) they specifically prohibit it but they don't say why....
Thanks!!!
In the first round of affinity selection from phage-displayed libraries you typically start with roughly 100 to 1000 copies of each clone, of which maybe only one (or a few at best) are retained by the target and get amplified for further selection rounds. Thus, applying negative selection to initial library is not considered productive as you risk losing even the best binders. After you amplify initially eluted phages, the input for the next selection cycle can be tens or hundreds of thousands of each clone that has "survived" the first round, and performing negative selection prior to contacting enriched library with the actual target effectively removes target-unrelated clones, while at least some of the relevant phages are retained. Hope this helps.
For a negative selection, it depends on your phage library. Is your bank naive or not? I do routinely phage display in my lab to isolate new binders or new target. We work with a naive phage library. We saw that a depletion step before selection gives always better results. I always do one or two depletions before the selection step, and at every rounds of phage display.
If your library is not naive, the depletion step might be less effective. In this case, it depends on your target.
Hope this helps.
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