protein purification & imidazole problems

[Dr Engelbert Buxbaum]

Am 30.03.2009, 04:34 Uhr, schrieb Chris McDermott-Roe  
<cjmcdermottroe from googlemail.com>:

> Hi all,
>
> I've expressed a recombinant protein in E.coli, purified it via Ni-NTA
> chromatography and am now attempting to measure its potential inhibitory
> activity in a fluorescence assay that involves excitation at 485 nm and
> measurement at 530 nm.  The problem is that my protein prep is giving a  
> huge
> signal (as is my buffer alone control) which makes me think the imidazole
> (which is present in the elution buffer) is fluorescing.

Test the components of your buffer singly and find out which is causing  
the signal. Also make sure you are not looking at stray light: sample  
should be filtered over a 0.22 µm low protein binding membrane filter,  
suitable high-pass filters in excitation and low-pass filters in emission  
beam.

Btw, before I'd use the protein preparation I'd probably do a buffer  
exchange. Although His does not fluoresce, in high concentrations it will  
affect buffering of your samples and also have a kosmotropic  
(water-ordering) effect that can seriously change your proteins  
properties. Gel filtration, dialysis or ultrafiltration are possibilities.