ELISA ALTERNATIVE - (Dec/11/2007 )

Hi
I have recently isolated peptides that bind to a target using phage display. I need to further confirm this binding by alternative methods. I have been trying elisas for some period now without any success. Does any one know of a viable alternative to elisa to confirm my phage born peptides are indeed binding to the selected target.

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Hi
can't you try gel filtration?

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hey. sorry for my ignorance but i thought gel filtration was for sorting differing sizes of proteins. I fail to see how this technique can help me confirm that two proteiens (ab+ag) are binding.

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Biacore, if you have the facility available. Should work in ELISA though I think.

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unfortunatly i dont have this facility

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hi
wat is the ELISA protocol?
if the problem is something to do with the confirmation then obiviously u donot see any better results with BIACORE.

sravan.

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Target suspended in 10mMNa borate and plates coated 2h/30c
Washed pbst
Blocked , 0.1M NaHCO3 pH8.6 +5mg/ml BSA
Washed PBST
Synthesised peptides (biotinylated) in 0.1M NaHCO3 pH8.6 +5mg/ml BSA diluted down the plate with a staring conc 1ug/ml. 2h/30c
Washed PBST
1:5000 HRP-streptavidin diluted in PBST and added to plates 1h/30c
Washed TBST
100ul TMB substrate added watch for colour change.

When i choose one target i do not see much colour change i.e no binding?
if i use a second target that i also raised peptides against i get a colour change but the controls also are the same colour. non specific binding?

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Not an expert, How about Co-IP (co-immunoprecipitation)?

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hi,

if applicable in your system, try a zero-length crosslinking/western blot procedure

Anal Biochem. 1990 Feb 15;185(1):131-5. Links
Zero-length crosslinking procedure with the use of active esters
Grabarek Z, Gergely J.
https://www.ncbi.nlm.nih.gov/sites/entrez?c...st_uids=2344038

Sebastien

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If the peptide binds to the protein of interest with any reazsonable level of affinity, the complex will migrate as a larger protein. To do these expts successfully, you'll need purified target and peptide. Run each component down the column separately, then as a complex (using the same amount of each component). If there's a decent interaction, you'll find the peak for peptide alone will reduce/disappear, and a new peak will appear.

You can also do some analytical ultracentrifugation to look at stoichiometry.

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