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Buffer fluorescence signal?!


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#1 claire84

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Posted 31 January 2013 - 09:12 AM

Hello everybody,
I'm doing some enzymatic kinetics using the HIV-1 aspartyl protease. In particular I'd like to determine the Km of the enzyme for a particular substrate (http://www.sigmaaldr...ng=en&region=GB) in order to decide which substrate concentration to use for my inhibition assays.
I'm using a MES buffer (25mM) with NaCl (200mM), 5% Glycerol, 5% DMSO, 0.0002% triton X-100 and 1mM DTT.

I should measure the increase of fluorescence due to the activity of the protease.

During the measurement I encoutered some problems regarding the signal of the blank (buffer+substrate). It's very high and noisy.
is it possible some of the buffer components show fluorescence at the same wavelenght of the substrate? (ex: 320nm, em:420nm)
Or maybe is the problem related to the stability of the signal?The wells where is the blank are the first that are measured....

Any suggestions??

Thanks a lot!

#2 bob1

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Posted 31 January 2013 - 11:49 AM

I was recently reading about problems with fluorescence readings and types of plate with EtBr, but I think that was more in the 600 nm range of emission. Triton has some fluorescence but absorbs in the 200 nm range and emits in the 300. Can't comment on the others though.

#3 claire84

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Posted 04 February 2013 - 03:31 PM

Maybe is due to the percentage of glycerol?




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