Phalloidin Alexa 647 vs Alexa 488 - (Feb/11/2014 )
I have been trying to image cells stained with phalloidin tagged with Alexa 647 using TIRF, but the signal is very low compared to cells stained with phalloidin with Alexa 488. I use the exact same protocol to prepare the cells in both cases, but for some reason, the Alexa 647 comes out very dim.
I have attached images for each below.
Any one have any ideas as to why this is?
There could be a few reasons - are the two phalloidin conjugates at the same concentration? Do you have the correct filters for the 647 on the microscope? Are the emission curves more or less the same height?