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.
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#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.
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bachai
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Posted 28 June 2009 - 07:01 PM
K.B., on Jun 25 2009, 05:44 PM, said:
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.
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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).
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.
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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
K.B., on Jul 3 2009, 04:22 AM, said:
That's strange... DOI links should work fine. Check your inbox - I've sent you this paper.
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.
