Affinity purification of Transcription Factor

bjkeating at my-deja.com bjkeating at my-deja.com
Thu Jul 13 12:24:42 EST 2000



Hello all,

I be really grateful if anyone could give feedback/advice on an unknown
transcription factor I've been trying to purify (for over 8 months and 5
ulcers!!).

I am trying to purify this unknown transcription factor using oligoduplex
attached to dynabeads. The transciption factor is thought to be about 30-
35 kDa from UV-cross-linking experiements. I am basically using a 'one-
step' purificaton approach as the oligoduplex-bound dyna beads are passed
over crude nuclear lysates (the recovery/activity after using a spectrum
of resins and columns has been pretty poor). While there is recovery of
the protein of interest (evident from EMSA's) there is a lot of other
unwanted protein that are also binding (this is evident from silver-
staining). My humble thinking is that they are simply protein that like
to stick to DNA, I have varied the poly dIdC and salt concentrations but
there is little effect.
As well as using non-specific and mutated oligoduplexes, a notion I had
was to anneal PEP-primers overnight to form some kind of random oligo-
duplexes and then to pre-incubate the nuclear lysate with this mixture
(PEP-primers are every combination of random 15-mer primers used to
amplify tiny amounts of gDNA - in theory it PCR amplifies a whole
genome).

The questions I have are what concentration of "PEP-duplexes do I pre-
incubate with? (the concentration of the specific oligoduplex normally
used is about 50 pmol) how long for? (Any feedback at all would be
appreciated).


Also, does anyone know of an Academic and/or a commerical service that
offers a MALDI-ToF Mass Spec. service, as the yield of this protein are
about 500fmol per 10 litres and it is extremely hard to scale it up.


  Thanking you,


   Brendan


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