Strange size of pET-22b expression product

Mikael Niku mikael.niku at nospam.vetmed.fi
Thu Feb 26 02:55:27 EST 2004


Thanks for the comments.

Yes, I'm doing reduced SDS-PAGE, so dimers should be out.
The calculated molecular weight including the pelB signal and the His tag is
about 10 kD. However, I guess the pelB signal should have been cleaved, as
I'm finding most of the protein in the periplasmic space and in the medium.

I'm quite a novice in SDS-PAGE... is it really possible (or common) that a
very polar sequence looks double the "actual" size?

In fact, I should also ask about handling the pelB signal. What would be the
easiest way to check if it has really been cleaved or not? So far I haven't
been able to find an antibody, or a peptidase to use, and with the larger
extracellular constructs the size difference is a bit small to be reliably
checked in SDS-PAGE.

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   Mikael Niku             URL: www.helsinki.fi/~mniku/
   University of Helsinki  Dept. Basic Veterinary Sciences
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