Dear scientists,
I am currently doing some researches on live HEK cell imaging using quantum-dots. I basically pre-conjugate a biotinylated-toxin to streptavidin-Quantum-dots (ratio is 3:1), and this toxin could later bind to a specific type of channel that was transfected and expressed on HEK cells. I then leave the conjugation for at least 30 minutes to let them bind, and then add this conjugate to live HEK cells for 1.5 minutes at room temperature, then do 7 washes. (the final concentration is 150pM-50pM)
But in Negative control which the cells are not transfected and there are no that type of channels on HEK cells, I could still see QD signal moving, so I assume there are un-specific bindings. I am struggling here, so what could I do to remove those unspecific bindings, should I just wash it more intensely? And how to avoid those fake round auto-fluorescence in the background when imaging?
Any comments would help.
Best Regards,
Hi Shiju,
Perhaps you have some unconjugated Q-Dots that are giving signal in your controls. Performing a mock experiment adding unconjugated Q-dots plus incubation/washing and verifying whether Q-dots in these cells look like those in your controls will tell what's going on. Then you'll have to find a way to exclude this unconjugated species. A exclusion column, dextran, may do the job.
Cheers,
i guess ...the selection of Q-dots must be again looked into....
for this experiment...may be you may choose QDs...with a narrow spectrum/ that must be different from the autofluorescence region...
also...in some cases if the QDs have bleached/lost fluorescence/old/damaged lot...you may not get significant change in control and test samples...in this case you may choose to replace with fresh lot of QDs and read the precautions/manual for handling QDs.
I hope this helps!! Let me know!!
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