I am interested in understanding the spatial dynamics of certain proteins with time in live neurons. Instead of creating fusion proteins, I was thinking of using quantum dots based conjugation of antibodies specific to these proteins and then following these proteins by the live cell imaging in these neurons. Actually using quantum dots of various colours (emission wavelengths), I can simultaneously track 2-3 proteins in neurons. This will help me to better understand the interactions of the target protein with other proteins.
I would be grateful if someone can share their experience of using quantum dots for live cell imaging of proteins. It will be a great help.
Hi Apurva,
Such methods have been used before. For example see:
http://cancerres.aacrjournals.org/content/67/3/1138.abstract
However, the following caveats apply:
1) Is the Ab known to be specific for your cell/protein of interest?
2) Does Qdot conjugation alter Ab affinity?
3) Does the Ab-epitope interaction alter function of your protein?
4) How long will the Ab binding last i.e. what is your experiment timecourse?
5) Will enough Qdot be conjugated to the Ab to allow adequate signal resolution in the abscence of a secondary amplification step?
Of course, the use of fusion proteins presents another set of complications as I'm sure you know!
BW,
David
@David: What would be better : to create fusion proteins or use quantum dots for the above mentioned purpose as per your experience?
It depends exactly on what you want to do. It's likely both methods will be required for novel studies since, taking the case of receptor trafficking as an example, you would: 1) use a transfected GFP-tagged receptor; 2) use a QD-labelled anti-receptor Ab to confirm that the approach employed in (1) is faithfully tracking the native receptor; 3) use a QD-labelled anti-receptor Ab to allow single protein visualisation at magnifications >60x. However, if you are simply looking at a protein with known dynamics, then QDs alone may be sufficient.
See https://www.sciencemag.org/content/320/5873/201.full for a good example of using QDs to track GluR mobility.
From my own experience, you should make sure that the Qdots are binding to the correct target (i.e. run all kinds of negative controls: Qdots with non specific IgG, etc). One relatively unpopular feature of Qdots is that they are sticky, and bind all kinds of non specific targets. I spent about 6 months optimizing conditions to follow Qdots using TIRF with only moderate success. The Qdots love the glass!