I want to do a fluorescence polarization experiment to look at the binding of avb3 integrin to an ELP nanoparticle that should have quite high affinity for it. While for this type of experiment the receptor is typically unlabeled, my thought is that the nanoparticle (abt. 35nm diameter) is much too large to be the "tumbling" component. This leaves me with labeling the integrin, which is available in 50ug quantities (and is quite expensive). I will need to buffer exchange the integrin before labeling (tris is not compatible with NHS chemistry) and will also need to remove free dye after the labeling reaction. This is likely to result in significant loss of integrin, and substantial increase in the cost of each experiment.
Does anyone have any ideas for setting this experiment up which may be more efficient than the above method?
No. But on the other hand if you label and use something like priceton separations 'centrispin' columns (1 should be sufficient) for getting rid of your excess dye then you get roughly 70% back in my hands. Which, if you label up all your protein should be ok for single-molecule studies.
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