Hello Everyone,
I was calculating kd of a 10 aa long peptide with one of my FL protein (MW 25kda, contaning 4 Tryptophane resudes) , It has two domains; one of the domain makes a dimer and other stays as a monomer, overall FL protien is making dimer and the peptide binds in between the dimer interface and peptide also has close contcat with one of the tryptophane out of the 4 tryptophane residues (1 peptide/2FL moleucles, litrature suggested).
1. I titrated my peptide using the range of 0.0025 nm to 500 nm with 1miroM FL protein and I got this graph, I have plotted this using one bindig site and two binding sites. please see attachment.
2. I also titrated peptide using the range of 0.1microM to 5 microM with the 1miroM FL protein and I got this graph, I have plotted this graph using one bindig site and two binding sites, please see attachment.
If you see both the graphs; using one binding site - graph does not fit very well, however, using two binding sites - graph fits very well. How can I confirmed using this data set whethere my protein has more than one binding site or is it a artifact of Try floresence method for kd in the range of nanoM?
Please let me know If you need further information
Thank you in advance.
Rohit
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