Loss fluorescence when clone a fluorescent sensor into a differenet vector - (Jun/10/2015 )
I tried to clone a fluorescent sensor into another vector. I added a linker SAGGSAGG and a flag tag like sequence between the targeting sequence and the sensor. But the new construct just loses fluorescence. What could be the reason behind?
Thanks a lot
I'm assuming you've sequenced the vector and found no point mutations, stop codons or frameshifts. If not I'd start there.
Beyond the DNA level though, could you provide more information about the sensor?
Is this a circularly permuted type of sensor (such as GCaMP)?
Are you sure you have expression via western blot or ICC?
Also, where are you targeting the sensor? Some sub-cellular compartments can impede fluorescence. Oxidizing environments such as the ER, or extreme pH environments such as in lysosomes are notorious for causing fluorescence problems. What is the pKa of the chromophore in your fluorescent sensor? What is the maturation time for the protein normally? These are all factors that might play into altered behavior when you change a fluorophore's localization in the cell.