EMSA with recombinant protein - (Dec/02/2006 )
Has anyone done EMSA with recombinant protein instead of nuclear extracts?
I am trying to figure out how much protein to use (how many ng or ug).
Any help would be greatly appreciated!
I'd say in the ng range would be OK say 50ng, 5ng, 0.5 ng per lane. If you look at the active motif website their 10-20ng of their recombinant standards are equivalent to 5-10µg of total nulcear extracts from stimulated cells.
It may also depend on whether your protein needs to oligomerise with itself to bind DNA and whether the recombinant form will do this spontaneously or does it need to be phosphorylated to be able to do this and whether it needs another protein to do this.
All the best,
I agree with the above post.
My experience is that anywhere from 10-250 ng
of recombinant protein is necessary.