Help Please Stop Flow Fluorescence sx-20 - (Aug/22/2011 )
Hi, I'm a grad student new to the forum.
I'm having trouble measuring a kon using stop flow fluorescence. I'm using a sx-20 Stop flow machine exciting at 280nm and measuring a intrinsic tryptophan quenching with a high pass filter after 320nm. The problem is that even with buffer only I'm seeing a decrease in fluorescence voltage. I'm wondering if this is photobleaching or some other problem.
I've already determined Kd and koff on a static fluorometer with reliable numbers and there is quite a large decrease in fluorescence at ~ 340nM. So I was thinking it would be easy to measure kon with the stop flow. I know I can get a estimate of kon using the Kd and koff but I would like to measure it directly.
Does anyone have any insight or experience with the sx-20 machine and this type of experiment and can offer some guidance.
Go through the checklist and follow manufacturer instructions. Air bubbles can do that. Did you align everything?
Update: It was photobleaching the problem was fixed with a fluroescent analog.