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.

Thanks Much

cw

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.