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Peptide dotblot and ELISA


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6 replies to this topic

#1 bachai

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Posted 24 June 2009 - 08:53 PM

Hi all,
I wonder if someone can tell me how to improve retaining of synthetic peptides on PVDF or nitrocelulose membranes for dotblots? I found a paper reporting dotblots failed with peptides (~20aa long) but direct coating ELISA seemed to work better. I would apperciate much if you can point me towards a good review on this issue. Thanks.

#2 K.B.

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Posted 25 June 2009 - 12:14 AM

Friend of mine was trying to do that with 5-7 AA peptides. Western-blot and dot-blot failed, but she managed to develop quite reliable ELISA for quantitative determination. She's using competitive ELISA with peptide conjugated to poly-lysine (with EDC) for coating.

#3 bachai

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Posted 28 June 2009 - 07:01 PM

Friend of mine was trying to do that with 5-7 AA peptides. Western-blot and dot-blot failed, but she managed to develop quite reliable ELISA for quantitative determination. She's using competitive ELISA with peptide conjugated to poly-lysine (with EDC) for coating.


Thankyou very much K.B. Your friend's peptides (5-7aa) were obviously too short for an immune assay so she had to couple them with a poly-lysin as carier molecule for ELISA. Can you tell me what EDC is? Did your friend published the method somewhere, or can she share the trick with me? Many thanks.

#4 K.B.

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Posted 30 June 2009 - 11:26 PM

EDC is a coupling agent, N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide, CAS Number 1892-57-5.

You can find brief description of preparation of immunization conjugate, antigen conjugate for ELISA and ELISA in: doi:10.1016/j.idairyj.2008.10.011 (contact me if you don't have an access).

#5 bachai

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Posted 01 July 2009 - 08:22 PM

Thanks K. B. The link indeed can not be opened from my computer.

#6 K.B.

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Posted 02 July 2009 - 10:52 AM

That's strange... DOI links should work fine. Check your inbox - I've sent you this paper.

#7 bachai

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Posted 15 July 2009 - 07:15 PM

That's strange... DOI links should work fine. Check your inbox - I've sent you this paper.

Thanks K.B. I've got it.
I've tried a 19-mer peptide which was both OK in direct coating ELISA (PolySorp plastics, carbonat buffer), and competition ELISA. The peptide concentrations in range 0.1-2mM gave strong and specific signal with Abs, yet did not appear to reach the saturation.




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