Hi,

I‘m having an issue with some ChIP experiments I have been doing with a GFP antibody and protein A dynabeads. I’m basically incubating overnight with my antibody and then using protein A dynabeads to pull down the following day. My problem is that when I switch from my binding buffer (50mM HEPES 7.5, 20 mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.1% sodium deoxycholate, protease inhibs) to my first wash buffer (50mM HEPES 7.4, 140 mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.01% sodium deoxycholate) the beads aggregate and clump together but only in my tagged strain and not in my untagged negative control. This persists through all of my washes (0.5M NaCl buffer and 250 mM LiCl buffer) but gets much better when I do the final wash in TE. All of my washes are done at 4 degrees and the buffers were kept on ice. This has repeated several times now.

My qPCR results don’t seem completely off but the background I’m getting at loci where my protein really shouldn’t be (euchromatic regions) is quite high. I think this is because the beads aren’t being washed adequately because of the aggregation.

The pic below shows the aggregation. left is my negative control (untagged IP), right is my tagged strain.

Has anyone else noticed anything like this with dynabeads and found a solution?

any suggestions welcome! Thanks in advance!