Glutathion OD readout fluctuates

[Michael Sullivan]

If you measure the UV absorption spectrum of your buffer (using a  
water blank, which should have no absorption), you'll see why you  
can't use your buffer as a blank. On my spec, the A at 280 nM of 0.2%  
triton X-100 is nearly 3 abs. units and a 10 mM solution of  
glutathione is beyond the limit of the instrument (~3 Abs. units).

Most spectrophotometers I've used can only measure 2-3 absorbance  
units over a blank with no absorbance. Any absorbance in your blank  
is effectively subtracted from this usable range, so if you're using  
a strongly absorbing buffer in your regions of interest, you've  
effectively reduced your usable range to nothing. Many specs manifest  
this with wild fluctuations- which I think is what you are seeing.

If you want to use spectroscopy to make these measurements, I think  
you'll need to use a different buffer, or exchange the buffer of your  
sample for something more UV friendly.

Hope this helps.

Mike

On Apr 19, 2007, at 4:22 AM, Josmar wrote:

> Dear All,
> trying to use 30mM glutathion as a blank in OD measurement seems
> impossible because at both wavelenths 220 and 280 the readout
> fluctuates heavily. This happens with pastic UVettes by Eppendorf
> (220-1600nm) in two different photometers.
> The other buffer components are: 50µM ZnAc, 50mM Tris pH 8, 150 mM
> NaCl, 0,2% Triton
> Any hints on why this is so are highly appreciated!
> Josmar
>
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)