DNA binding proteins - help (Aug/14/2006 )
Hai all
I am purifying DNA binding proteins using PEI 1% to enrich DNA binding proteins that bind to a target DNA. I am using promega streptavidin paramagnetic particle to bind my 500bp amplified DNA with biotin at one end.After i bind the PEI precipitated to tne DNA containing beads,i elute with 1 M KCl , I am unable to get rid of lot of non specific binding proteins that come along with the real ones..please suggest a way to minimize non specific binding.
Thanks in advance
I am purifying DNA binding proteins using PEI 1% to enrich DNA binding proteins that bind to a target DNA. I am using promega streptavidin paramagnetic particle to bind my 500bp amplified DNA with biotin at one end.After i bind the PEI precipitated to tne DNA containing beads,i elute with 1 M KCl , I am unable to get rid of lot of non specific binding proteins that come along with the real ones..please suggest a way to minimize non specific binding.
Thanks in advance
Do you have a gel image of the protein mix? How many "non-specific binders" do you think you have?
The non-specific proteins - are you sure they aren't co-regulators of the DNA-binders (Could be interesting)? You might be able to get rid of true non-specific binders with a bit of detergent like Triton or Tween (0.01% to 0.1% for a starting range)
PEI is a very nasty molecule that I would suggest you not to use if you have other options. Under the condition that you have used, there will be extra free PEI in your crude sample. Free PEI will bind your DNA sequence and block specific interaction between protein factor and your target DNA.