Non-radioactive EMSA - (May/19/2008 )
I am currently looking for non-radioactive EMSA method which applicable to my work. My department here discourage us to use the radioactive way as it was too complicated in term of waste disposal. My work is different as I will be using in vitro translated protein with biotin tag(TNT kit & trascend tRNA,promega). I found a non-isotopic EMSA kit from Invitrogen which stain the protein with SYBR red. I wonder would it be possible for my biotihylated protein to detect by SYBR red? I couldn't remove the biotin tag as it wasn't in vivo recombinant protein. Hope that anyone here can help me with these, thanks.
If you have a biotin tag, you might consider a luminescent assay, with either streptavidin-alkaline phosphatase or streptavidin-horse radish peroxidase, followed by detection of the enzymatic activity. Check the Pierce web site (or Roche). This would be far more sensitive than sybr red, but you might not need the sensitivity.