[RASMB] AUC capabilities

[Ewa Folta-Stogniew]

Borris,

thanks. I discarded the labelling approaches for the reasons you mentioned.

At 25 nM the measured A280 in buffer of chose is 0.018; A220 =0.1.

Ewa

At 07:46 AM 1/27/2011, Borries Demeler wrote:
> > >2. If you use absorbance, there is a question about the extinction
> > >coefficient and the wavelength. Depending on extinction, 25 nM may
> > >be well within reach at several detection wavelengths.
> >
> > A280(0.1%)=0.85  (mg-1mL cm-1)
>
>Ewa,
>
>if my math is correct that's rouhgly 17 micromolar for a 59 kDa protein.
>Ideally, you want to measure from 10 fold above to 10 fold below 25 nM.
>At 25 nM your OD at 280 nm would be way too low to get any reasonable
>signal. So the answer is no, it is unlikely you will get reliable data.
>
>You may want to check how much extinction you will gain by going to
>a lower wavelengths, say 215-220 nm, but I doubt it will be enough
>to get into the neighborhood of 25 nM. Also, then you MUST work in a
>non-absorbing buffer like low conc. PO4, plus a bit of NaCl.  In my
>opinion this is not doable at 280 nm.
>
>Your best bet is to end-label the protein with a fluorophore (Alexa 488
>would be good) and then measure using an XLA with the Aviv FDS attachment
>at 3 concentrations, 1 nM, 25 nM, and 250 nM. This would give you your
>best shot at obtaining some useful results. There are gotchas with that
>as well, for one, you will need a very accurate assessment of labeling
>efficiency if you want to trust your concentration measurements, and
>you need to be sure the fluorophore doesn't interfere with dimerization,
>otherwise you change the very thing you want to measure.
>
>Regards, -Borries