The assay will be used to capture transcription factors with biotinylated oligos bound to magnetic beads. The proteins will be then identified with mass spectrometry.
There are several protocols doe DNA-protein pulldown out there that differ (considerably) by:
- oligonucleotide length and concentration
- controls used (just empty beads, DNA competitors, unrelated DNA)
- amound of protein used
What in your experience works best?
What is the minimal oligo concentration / length that can be used for this?
Are there any possibly tricky parts of this experiment that you could give hints for?
I will appreciate any practical comments. Thanks!
A lot of this will depend on the system you're studing. How large are footprints of similar TFs? Is your cell lysate expressing an active version of the TF? How strong is the binding affinity?
From my perspective, it is good news that you are studying a developmental/multicellular system. It seems that multicellular organisms need TFs with strong binding affinity so their impact endures the entire developmental phase. Bacteria live by a much shorter stimulus-response-reset cycle. A response that far outlasts the stimulus can be just as detrimental as no response at all.
Send me personal message if you'd like to discuss details. Seems like an interesting system.
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