[RASMB] AUC capabilities

[Brian Baker]

Hi Ewa,
This is going to be a tough experiment by AUC, ITC, or SPR. Have you
considered flurorescence anisotropy? Label em up, then simply start
diluting. You should be able to get down into subnanomolar concentration
levels with fluoroscein and a good detector.
On Jan 27, 2011 8:16 AM, "Ewa Folta-Stogniew" <ef55 at email.med.yale.edu>
wrote:
> 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
>
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