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I'm working from google translate (I can't read German), so apologies if I misunderstand some of the questions.
1) For my ELISA I am not using ab94898, I mention that only to show that the standard is still good.
2) I performed another dot blot today using a new standard (recombinant hEGF from Gibco, sequence NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR) and new loading buffer.
In this dot blot I could detect the native protein at 10ng, although the signal was quite weak. I still could not detect either denatured nor denatured and reduced protein (up to 40ng, film exposed for 15 minutes).
It seems that detection for the denatured state is very poor. I suspected that it might be due to proteolytic cleavage (even though the sample is not in a crude tissue lysis), however previous attempts with added protease inhibitors in the sample buffer made no difference.
Hope this helps,
Asked on Dec 13 2012
Thank you for your email and the great effort to translate everything into English. I am very sorry, I confused the languages in your email and wrote you in German. I hope you will have it easier to read this email.
Thank you very much for confirming the protein sequence. That shows that this antibody is detecting the native protein, as is also your ELISA experiment.
I contacted the lab to get some confirmation if it was observed before that this antibody has problems to detect the denatured protein.
I am apologise for the inconvenience you had perform the dot blot with this antibody and would like to offer you some compensation in this case.
We offer four other antibodies against EGF, which are covered by our guarantee for WB and human as follows.
1. Anti-EGF antibody (ab9695)
2. Anti-EGF antibody [1B11-3E12] (ab40644)
3. Anti-EGF antibody [EGF-2] (ab8282)
4. Anti-EGF antibody (Biotin) (ab84263)
If one of these suits your experiment I would be pleased to send you a free of charge replacement of one of them. If none of this antibodies full fills your needs I would also be pleased to send you a different antibody as replacement or to give you a credit note.
Thank you very much for your cooperation. I look forward hearing from you how you would like to proceed.
Answered on Dec 13 2012