Hi everyone

I work on the structural analysis of a small DNA-protein complex (~150 KDa) using cryoEM. After reconstruction of this complex from purified proteins and DNA (42 bp), the complex tends to dissociate upon gel filtration and during the grid-freezing process. We have the idea to crosslink it, but I have some difficulties. I tried different conditions with formaldehyde and glutaraldehyde (with and without using Grafix method) and I obtained crosslinked protein complexes but without DNA. Furthemore when the proteins are crosslinked, its don´t bind DNA anymore (I can see that with native gel analysis stained with Coomassie and Toluidine blues to reveal protein or DNA respectively). Do you have any suggestions to perform the crosslinking of DNA-protein interaction ? (I know this is done for CHIPseq, but in cellular extract)

My starter buffer is 10 mM HEPES pH8, 120 mM KCl, 4 mM MgCl2 and 1 mM TCEP

Thanks in advance