Dear All:
I have a his-tagged transcription factor that I wish to purify using
TALON magnetic beads. Ulimately, I want to conduct the SAAB assay
(selection and amplification binding); briefly, one incubates the
protein with a degenerate oligomer of 60 nt (20nts on both ends of
defined sequences and 20 N's in the middle). If there is sequence
specific binding, one could 'wash off' all unbound oligomers and then
PCR the 'bound' DNA to amplify sequences that are capable of binding.
This material then is used for subsequent rounds of binding and
amplification and in the end, one clones the oligomers, sequences them,
and hopefully arrives at a consensus binding site for the transcription
factor.
I'll admit to being lazy in trying to save steps but my real wish is to
keep my his-tagged protein happy and healthy. My idea is to bind my
protein to the TALON beads, then directly incubate with the oligomers
instead of first eluting the protein (which will require either
imidazole or low pH and which may interfere with DNA binding unless one
further insults the protein by dialysis, etc). Most SAAB protocols
employ EDTA and DTT in the binding buffer, two reagents which are
incompatible with the his tag binding to TALON (or at least the
instruction manuals lead me to be to believe that). Now for my
question; has anyone tried what I am proposing and or does anyone have
any experience with TALON beads to offer advice about how to streamline
this experiment? I've asked Invitrogen for a list of incompatible
reagents but have yet to get a response.
Thanks for your time
Dan Riggs
