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phage display -peptide differentiation

phage display -peptide differentiation - Peptide Forum

phage display -peptide differentiation - Peptide Forum. Ask and discuss questions on peptide protocols, custom peptide synthesis, peptide identification and peptide sequencing.


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  #1  
Old 07-27-2010, 12:42 PM
Pipette Filler
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Default phage display -peptide differentiation



I'm working with phage display libraries from NEB. I want to find a non destructive way of differentiation between/grouping peptides from a sample. Each phage has between 3-5 identical peptides displayed on its coat protein. Each sample (0,5-1 ml of eluted phage) contains 10^2-10^4 (estimate) phage particles, where the phage part is the same, only difference is the peptide. There could be up to ~50 clones of each peptides-specific phage.
Any ideas as how to do this?
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Old 07-29-2010, 12:39 AM
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Default Re: phage display -peptide differentiation

The NEB PhD libraries have the lacZα-peptide cloning sequence so you can do blue white screening. It gives you phage plaques including individual phage particles so you can isolate individual particles and sequence the vector to identify the sequence of the peptide displayed on that phage particle. I believe these protocols are included in the NEB manuals.
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Old 07-29-2010, 07:44 AM
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Default Re: phage display -peptide differentiation

Yes I do know the protocol for phage display...
I am talking before plaque selection/plating the phage. Besides I'd have to send all the plaques in for sequencing to find out how many different clones there are, and since I want to do this with no prior knowledge of the sequences that would be pointless.
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Old 11-30-2010, 05:05 PM
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Default Re: phage display -peptide differentiation

I am performing phage display also. I have a problem. As all or 99 % from the peptides I got after the 3rd round are the same, the same phage, as if this library (7 mer or 12 mer from NEB) only contains this phage. That means only one phage clone dominating the phage pool. And the other peptides (if any) they are not that similar to the dominating peptide, making it very difficult to derive a consensus sequence.
When I wanted to test the relative binding affinity of this peptide which dominates the phage pool by phage Elisa (using anti M13 Abs), the OD at 405 nm is less than 1.

Thank you very much for you helps.
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Old 12-02-2010, 06:50 AM
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Default Re: phage display -peptide differentiation

If you explain more in detail your protocol it might be easier to help...also which target you are using to isolate the peptides (proteins, antibodies, cells etc.?).

Did you check if your anti M13 Ab is still good? Is it conjugated, maybe the conjugate is off? maybe the substrate? What is the relative OD you get from a KE phage?
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Old 12-02-2010, 10:52 AM
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Default Re: phage display -peptide differentiation

Thank you very much for your reply

my target is a protein (enzyme), I am searching for a binding peptide for.
I am working with phage display protocol from NEB. M13 Ab are HRP conjugate, I think it is ok, because I see very week ELISA signal ( or sometimes no signal at all) for the target uncoated well on the ELISA plate. The substrate ABTS is new.
If I understood you correctly, for 109 pfu per well, I get 0.8 OD at 405nm maximum and some time less.
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Old 12-02-2010, 03:29 PM
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Default Re: phage display -peptide differentiation

well if you get the same signal using your clone as when using the KE phage (without peptides), then you could try to capture the phage with unconjugated anti M13.
You could also try using the unconjugated anti M13, and then using a secondary anti mouse AP conjugate, maybe that might give a stronger signal.

I would check the sequence you got for unwanted specificity. G.P.Smith published an article within the past few months, at most, reporting sequences of phage clones that aren't specific, they just grow faster than most others and thereby manage to dominate after a few rounds of panning.
An asian group also reported some sequences that were either found to bind to the plastic or other unwanted targets.
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  #8  
Old 01-20-2011, 06:54 AM
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Default Re: phage display -peptide differentiation

If you're having troubles with the solution, why not use a peptide calculator?
It can help measure the right amount to use..

Last edited by admin; 01-20-2011 at 06:15 PM.
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Old 05-03-2011, 02:42 AM
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Default Re: phage display -peptide differentiation

wow! this forum was great!. this thread is all i need to know because of the replies of the other users I learned all about all the difference of peptide by having ideas posted here... its great to see this forum... i learned alot! thanks for the posts!...
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