2 replies to this topic

[cwilsonbran]

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

[gorkin]

Go through the checklist and follow manufacturer instructions. Air bubbles can do that. Did you align everything?

[cwilsonbran]

Update: It was photobleaching the problem was fixed with a fluroescent analog.