Glutathion OD readout fluctuates
(by mlsulliv from wisc.edu)
Thu Apr 19 15:32:15 EST 2007
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.
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!
> Methods mailing list
> Methods from net.bio.net
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
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