I am trying to develop a method to define protein binding to molecularly imprinted polymers. The method in brief: I am labelling my protein with FITC and then I add my MIP, and during incubation I check the anisotropy change. I can use only a basic platereader for the experiment, so I measure steady state anisotropy. With other methods (BCA assay kit) I can measure that my protein binds to the MIP. Unfortunately I can't see any anisotropy changes in my experiment. I have some ideas what could be the problem, but first I would like to ask your suggestions what the problem is. I have these theories now:
1. The FITC is flexibly attached to the protein, and I just measure the rotation of the fluorophore.
2. Because of the small Stokes-shift of fluorescein it undergoes self-quenching and that's why I can't see a change in anisotropy.
Do you think these are the problems?
Thank you for your answers in advance.
Your theory #1 is plausible. This propeller effect is a pretty common occurrence. You may be able to overcome it by attaching the fluorophore by a shorter linker.
Another reason for a lack of anisotropy change is a mismatch between the fluorescence lifetime and the molecular weight change upon binding. If the fluorophore is attached to a large protein, the anisotropy may already be as high as it is going to get, with or without binding to the MIP. You may need to change to a fluorophore with a much longer lifetime. The relationship between polarization, molecular weight, and fluorescence lifetime is explained here:
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/fluorescence-polarization-fp.html
Thank You for your answer! If I proceed with my work, I will inform you!
A bit mire information would be useful. As Adam says the MW of the protein is critical - what is yours? Also the labeling ratio is important - how exactly did you label the protein and separate the free probe. What is the labeling ratio? I am attaching a chapter on polarization which may help. Finally are you sure that your platereader is working correctly? Did you test it with - for example - FITC in alkaline buffer (which should be about 0.02 polarization) and in glycerol (which should be about 0.45)
Dear Mr. Jameson,
My protein is lysozyme MW around 14.3 kDa. I labelled it with fluorescein, and purified it with dialysis with a membrane that have a cut off 3-5 kDa. I measured the labelling ratio with an UV-VIS spectrofotometer. I measured the absorbance of the conjugate at 280 nm and than at 494 nm. I used the correlation factor that i found in a protocol which I found on Thermo Scientific page. I measured around 2,6 dye/protein ratio. I didn't test the platereader yet, but others in my lab who use it for fluorescence polarization don't have problem with it. Thank you for your pdf and I will inform you if any proceed is occured.
Hi Mirko,
Actually I have done the polarization of FITC labeled lysozyme. I got about 0.22. But I was careful to have it lightly labeled - about 0.5 probe to protein on average. Your labeling ratio of 2.6 means that about two thirds of your protein have 2 or more probes bound. So this would definitely lead to lower polarization due to homotransfer between the bound probes. You may want to try a lighter labeling. Good luck!
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