I am been working for a couple of months without success on setting up an assay based on GTPases loading with bodipy gdp and then measure the exchange to GTP in presence of various GEFs.
Reading on literature, the idea is that once the Bodipy GDP is loaded onto the GTPase, there is a significant increase in fluorescence (compared to Bodipy GDP alone) , which decreases when adding GEFs, which exchange it to GTP and thus releasing the Bodipy GDP.
I have been stuck on the first step, because after incubating my GTPase with Bodipy GDP I saw that there was no difference in fluorescence compared to bodipy gdp alone.
Among different protocols that didnt work, here is one: Article In vitro fluorescence assay to measure GDP/GTP exchange of g...
I store my GTPases in a simple Tris based buffer, tried to buffer exchange them into HEPES buffer that the paper uses but nothing. There are other assays that instead of Hepes use Tris, I have tried those too but nothing.
i think my GTPases cannot load the GDP for some reason and i dont understand why.
If anybody had been through this assay and would like to share any tip in protein storage handling or the assay i would be grateful.
thank you!
If the proteins already have GDP or GTP bound, then you may not be able to get Bodipy-GDP to bind, unless you simultaneously include an exchange factor.
Another thought is that if Bodipy fluorescence intensity is not affected by binding, try measuring fluorescence polarization instead.
Dear Dr. Shapiro, thank you so much for the reply. I should have mentioned the other protocols that I have used I am sorry. I have also tried bodipy GDP loading in RhoA and Rac1 in presence of EDTA and/or GEFs but there was no difference with bodipy GDP only. Which is why I am doubting whether it’s the protein which has some issues. As for the polarization , I will try that, do you think it could make a difference?
I have had success in some cases using polarization/anisotropy to measure binding of Bodipy-labeled ligands to proteins. Other fluorescent dyes can also be useful, such as tetramethylcarboxyrhodamine (TAMRA).
Attaching a large fluorescent dye like Bodipy can alter the ability of the ligand to bind, or its affinity. You should also try other fluorescent probes, if they are available.
Finally, I suggest you analyze the purity of your supply of Bodipy-GDP. Commercially supplied probes are not always of the highest quality. You may be able to find a TLC or HPLC method in the literature. It may be possible to repurify the supply using some sort of chromatography. I did this with Bodipy-penicillin using a column of Sephadex LH-20.
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