tan at aeolus.vmsmail.ethz.ch
Fri Mar 11 04:25:39 EST 1994
In article <2lnhaf$7gg at mercury.hgmp.mrc.ac.uk> lfernand at crc.ac.uk (Miss L.M.
>has anyone in the newsgroup used UV crosslinking
>to characterise the size of DNA binding proteins
>of interest ? a couple of the methods I've read
>uses micrococcal nuclease together with DNase I
>to digest unbound nucleotides after crosslinking...
>other groups don't use m.nuclease ... do i need this
>... if yes, where can I get it as I cannot find it in any of the
>catalogues I have
>thanks in advance.
>e-mail address : L.M.Fernandez at qmw.ac.uk
It's certainly not one of my favorite methods, but I have used UV crosslinking
to determine the size of DNA binding protein fragments. The main reason why
both DNaseI and micrococcal nuclease are often used is that neither enzyme is
truly nonspecific. For instance, DNaseI cuts poorly in very AT or very GC rich
regions. Combination of different nucleases often helps to reduce the number
of bases in the cross-linked protein-DNA complex, which leads to a more
accurate measure of the protein size on an SDS-PAGE gel. But DNaseI and
micrococcal nuclease need not be the best combination for your experiments.
For example, I used DNaseI and the restriction enzyme RsaI since the DNA piece
I used had a RsaI site just outside the binding site. Useful to try several
experiments with different nucleases, although DNaseI alone or DNaseI +
microccocal nuclease is a good start. One other point: the cross-linking
efficiency (one of the major determinants of the sensitivity of the method)
improves significantly if you incorporate bromo-dU into the DNA. I was unable
to get a decent signal without bromo-dU incorporation. Gory details as well as
a couple of references can be found in a paper of mine: Tan and Richmond, DNA
binding induced conformational change of the yeast transcriptional activator
PRTF, Cell, 1990, 62:367-377.
WRT source of microccocal nuclease, it should be available from distributors
such as Sigma, Worthington enzymes and Fluka. It's also known as
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: tan at aeolus.vmsmail.ethz.ch
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