I want to pull the transcription factors which binds to the biotinylated DNA probe of promoter sequence flanked 5' of a gene of interest. Any technical advice on the following questions?
Probe design
1) Any experience of using a relative long probe up to 2kb?
2) What factors to consider while choosing among various biotinylation methods (e.g. biotinylated primer, biotinylation kit)?
Preparation of cell lysate
1) To prevent the nuclease digestion of the DNA probe, adding chelator of divalent ions (e.g. Mg2+, Ca2+ ) is an option. Since the binding of some transcription factor may require metal cofactors (e.g. Zn2+), any suggestions on a non-chelator type DNase inhibitor?
(As an alternative, have considered to use a chelator preferential to complex with Mg2+ and Ca2+, for example NTPO from https://www.dojindo.com/Images/Product%20Photo/Chelate_Table_of_Stability_Constants.pdf but since DNase could be activated by divalent ions other than Mg+ and Ca2+, may not be a good option?)
Binding
1) Is it feasible to avoid secondary structure of the DNA probe by adding DMSO which flattens DNA?
Thank you!
Hi Johnny,
I haven't done such experiments by myself, but I talked to people routinely doing similar pulldowns with biotinylated DNA probes for transcription factors isolation, which works quite well.
Here is one paper from this research group as a starting point for you:
Article The CHR promoter element controls cell cycle-dependent gene ...
Hi Johnny Tam ,
Have managed to do assay?
Have you got a protocol?
Thanks a lot,
Marcelo.
I am also planning for a similar experiment, Can someone share the protocol
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