protein-DNA binding - how to measure a binding constant? (Jun/17/2004 )
We are embarrassed by measuring the binding constant of a transcriptional factor and its corresponding DNA sequence (28bp). Preliminary results from EMSA experiment showed that the binding may be weak since the mobility of the DNA fragment can only be shifted in the presence of hugely excess of the protein. We tried to measure the thermodynamic binding constant with ITC, but failed, because ITC only works well in cases with high degree of affinity. If someone could suggest any fast, sensitive and reliable techniques for our purpose, we would be highly appreciated.
Thanks in advance
Ph.D. Center for structural biochemistry
Novum S-141 57, Huddinge
You might want to have a look at fluorescence anisotropy. We have used the technique a couple of times in the past with good results (that actually correlate well with EMSA data). A more crude method you could use is circular dichroism spectroscopy. There are a number of papers out there documenting these interactions. A good start would be some papers on GCN4. Good luck