Some proteins are present in human plasma in both free and bound form. In most cases the free fraction exhibits (co)factor activity. However, the underlying mechanism responsible for binding, and consequently separation of both protein and ligand remains largely unknown. Therefore, I wanted to know if someone knows a way to analyse/investigate such a process. In this case, I am mainly interested in understanding the triggers.
Dear Rene,
you could employ MicroScale Thermophoresis (MST). This technique allows you to determine binding affinites indendent of your buffer system; you could measure even in serum or plasma!
One of your interaction partner needs to be fluorescently labeled for detection. The ligand is added in a dilution series around the expected dissociation constant. Now you can investigate the interaction in absence and presence of your co-factor and see how binding is affected. In a next step you could do the same in serum or plasma.
Here is a publication where MST was employed to investigate binding of a protein to a phospholipid in dendence of Ca2+-Ions.
http://www.nanotemper-technologies.com/technology/publications/article/jbc-the-journal-of-biological-chemistry-1/
And here you will find several publications where the binding affinity was determined in complex bioloquids:
http://www.nanotemper-technologies.com/applications/purification-free/
If you have any quesitons, please do not hesitate to contact me ([email protected])!
Best,
Heidi
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