E280 Protein quantitation on microplates ?!

Dr E. Buxbaum EB15 at le.ac.uk
Wed Mar 11 16:57:05 EST 1998


Marco Bocola wrote:
> 
> We build up a new biochemical workgroup and looking foorward to by an
> UV/VIS-Microplate Reader.
> Has anyone experience with UV-Microplates and especially Protein
> determination ?
> (Unfortunately we have no "normal" UV/VIS Photometer)
> Is it possible to measure quantitative absorption with good accuracy for
> small volumes?

The first problem here is not the reader, but the plate, because normal
96 well plates are made of polystyrene, which has considerable (read:
almost total) absorbance at 280 nm. Even if you could get plates made of
polyacryl, the absorbance probably will be some 50 or 60%. Additionally,
I do not know of a 96 well photometer which were able to produce light
of such short a wavelength (you need a mercury or xenon arc bulb, not
the normal tungsten filament lamps). You should be able to get that
information from the specs of the instrument you want to buy, however. 

To measure A280 for small samples, we use a submicro quarz cuvette
together with our Beckman DU-65 spectrophotometer. The minimal volume
with this setup is 50 ul, the cuvette can acomodate up to 100 ul, the
pathlength is 1 cm as for normal cuvettes. We use a "crystall" tip
connected to a vacuum line to empty the cuvette between readings, the
cuvette remains in the instrument. The speed is of course lower than
with 96 well plates, but you can still read 3-4 samples per min if you
are experienced. This system works very well for Warburg protein
assays.



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