[Protein-analysis] TALON/transcription factor SAAB assay

Dan Riggs via proteins%40net.bio.net (by riggs At utsc.utoronto.ca)
Mon Jan 8 11:36:49 EST 2007


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



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