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Customer has performed the Dot-Blotprotocolas suggested;please findthe results attached.
Regarding the storage:
- antibodies were stored at -20°C.
- recombinant proteins were reconstituted with 100 ul PBS + 15% glycerol, aliquoted andthen stored at -80°C.
Regarding the WB protocol, she said shealways do the ponceau stain to confirm the transfer, and her transfer buffer does not contain SDS.
Thank you for you support.
I am looking forward to hearing from you.
Asked on Jan 19 2012
Thank you for sending along the dot blots and additional information. We really appreciate the extra troubleshootingeffort.
Here are my thoughts:
1. In general it looks like the antibodies are able to detect Defensin in the chorioamniotic membrane samples.
2. It does not appear that the antibodies detected recombinant Defensin protein. You note the mass of recombinant protein spotted on the membranes, from 400ng - 0.78ng. Was the concentration of the Defensin proteinconfirmed by Bradford assay or OD280n measurements following reconstitution or are these number based on the expected concentrations after reconstitution?
If the concentrations are only theoretical, could you please check the concentrations to ensurethey are accurate?
Thanks again for your troubleshooting efforts. I'm sure we'll be able to resolve this situation soon.
Answered on Jan 19 2012