[RASMB] AUC capabilities

[Leech, AP]

Hi Ewa,

I would be sceptical of that measurement. It corresponds to a protein
extinction coefficient of 720000 M-1cm-1 (or about 12 A280/(mg/mL) )
which is a lot for a 59 kDa protein! Absorbance differences of that
order can happen just by moving cuvettes or spectrophotometer warm-up
drift.

Andrew

On 27/01/2011 13:16, Ewa Folta-Stogniew 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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Dr Andrew Leech                   *  Laboratory Head
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