I am trying to carry out pull down assay using magnetic beads with single stranded biotinylated DNA of interest. Image attached. Lane 1: marker
Lane 2: Beads only
Lane 3: Protein + Biotin DNA + Beads
Lane 5: Protein + Vehicle control
Lane 7: Protein + Non-biotinylated DNA + Beads
Lane 9: Protein + Biotinylated Scrambled DNA + beads
Lane 10: Just pure protein
I am getting bands in all of them irrespective if the magnetic beads were used or not. Could someone kindly advise me on this.
Hi Aruna, magnetic beads by themselves bind protein non-specifically.
Article Isolation of Low Abundance Proteins and Cells Using Buoyant ...
If you are using pure protein for the pull down assays using mag beads then you are setting yourself up for false positives. Try the pull down assay in the presence of non-specific proteins like BSA.
Hi Aruna,
you may want to check for the size of the band. It could simply be the avidin / streptavidin immobilized to the beads to capture your biotinylated DNA. It is rather common that your capture ligand "elutes" from the column / bead especially if you boil it in SDS loading buffer. Avidin / streptavidin are tetramers, so not each subunit is covalently coupled to the beads and thus if you denature the sample (i.e. boil and reduce) you'll elute those. This is generally not a problem as you'll have the same band in the negative control.
Cheers Markus
I tried BSA as the non specific protein, issue still remains, getting band in all. Kindly advise.
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