Peptide dotblot and ELISA
Posted 24 June 2009 - 08:53 PM
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.
Posted 25 June 2009 - 12:14 AM
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.
Posted 30 June 2009 - 11:26 PM
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).
Posted 01 July 2009 - 08:22 PM
Posted 02 July 2009 - 10:52 AM
Posted 15 July 2009 - 07:15 PM
Thanks K.B. I've got it.
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.