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
It's visually obvious that there are 2 very different binding affinities, so it is appropriate to fit the data to two different binding sites. Since the Kd of the higher affinity site (
Thank you Adam for the response,
I did not pay attantion to the y-axis scale.
I repeated two time uisng low range peptide , both the times I got the same result. I am also very sure that the peptide and protein concentrations are correct.
Is it possible that my peptide binds non-specifically (any suggetion to confirm this would be very helpfull) to the protein and that is giving the false saturation at 100nm? If it is corrcet what equaation should I use for fitting the graph?
If you have any suggetions for setting-up a new experimen please let me know.
Thank you
Non-specific binding is, of course, always a possibility. However, it is likely to be of relatively low affinity and is more likely to account for the higher Kd binding.
Another possibility is that not all of your protein is in a state that is capable of binding the peptide. That would account for the titration reaching saturation at a concentration that is much lower than the protein concentration. Some of the protein may be "dead" as far as binding is concerned.
Dear Rohit,
I'm also determining the Kd1 and Kd2 of my enzyme, which was proven to be a protein with two binding sites according its crystal complexes, by tryptophan fluorescence quenching method. However, I was wondering how can you fit your plots according to the two-binding site model. If possible, could you tell me the software or the equations I should use to process my data?
Best wishes,
Yu-Cong
Hi Yu,
I used prism-9 using this equestion:
Site1=BmaxHi*X/(KdHi+X)
Site2=BmaxLo*X/(KdLo+X)
Y=Site1+Site2
I hope it will work for you
Thank you
Rohit
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs