Advice on Southwestern blot?
ewlab at gandalf.bio.uci.edu
Thu Jun 1 15:56:46 EST 1995
I am going to try to screen an expression library (lambda gt11) by SW blot. I
have the conditions worked out using crude HeLa nuclear extracts and I do get
a single band corresponding to the MW determined by UV X-linking. Before I
embark on this venture, I am seeking some advice on the following questions:
1. What type of nonspecific DNA should I use? I have been using
poly(dI-dC). Some protocols suggest poly(dI-dC), others say to not use it
and use calf thymus DNA. What is it about poly(dI-dC) that would lead to high
background and/or false positives vs. other nonspecific DNA?
2. What type of probe to use? I have been using annealed oligos containing a
single binding site for the protein as well as a multimerized probe with
equal success. Will multimerizing necessarily enhance the signal for
bacterially expressed proteins?
3. How can you reduce false positives (ie nonspecific DNA binding proteins)?
4. Is it possible to show specificity or reducing false positives by doing a
competitive SW? (ie including excess cold self or excess ds oligo
containing mutated binding site)
Any advice on this matter would be appreciated. Thanks
mdpetros at uci.edu
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