protein-DNA interaction in gel filtration - (Dec/10/2007 )
I'm trying to adress interaction between two yeast proteins using gel filtration chromatography.
It seems that one of my protein is strongly bound to chromatin, and it masks the possible interaction with the other protein.
I have previously treated my whole cell yeast extract with 1mg/ml of ethidium bromide. But, this is not sufficient to remove the interaction of my protein with DNA.
I can not use DNase I because I must keep the extract at 4°C.
Do you have any suggestion ?
Out of so many methods that are suitable for studying protein-protein interaction, why do you choose this? not to mention this assay need a lot of proteins, what if the proteins dissociate from each other during the run?
I think I didn't explain well what I'm trying to adress.
Using gel filtration, I want to see the reorganisation of protein complexes following DNA damage treatment. Using non denaturing condition, the complexes should be maintained even during the running of the colomn.
Sorry for the misunderstanding.
Affinity capture followed by SDS-PAGE wont work in this case?
It seems that one of my protein is strongly bound to chromatin, and it masks the possible interaction with the other protein.
I have previously treated my whole cell yeast extract with 1mg/ml of ethidium bromide. But, this is not sufficient to remove the interaction of my protein with DNA.
I can not use DNase I because I must keep the extract at 4°C.
Do you have any suggestion ?
why not try increase the concentration of saline, which can strip the protein from DNA. also, in high salt the chromatin often condesated and can be easily removed, then the clean protein can be diluted to appropriated salt cons for interaction.