[FDS] Alexa 488
Tom Laue
Tom.Laue at unh.edu
Sun Dec 3 12:29:35 CST 2006
Hi all-
Sorry for not weighing in on this earlier... a grant is due tomorrow. Ugh...
I would like to add a couple of thoughts. Aside from its slightly better
extinction coefficient, Alex488 does not exhibit as much photobleaching,
and is far less sensitive to pH.
The quantity of dye needed will depend on whether you are conducting
equilibrium or velocity sedimentation. For equilibrium, 1 to 10 nM
Alexa488 would be needed, whereas about 100 pM is needed for velocity
(due to the data 'filtering' available from Lamm equation fitting) to
give you a S/N ~100.
To get to low dye levels the following is recommended:
1. Degas your sample. Molecular oxygen is a terrific collisional
quencher. I have seen signal levels improve 10-fold with
degassing. To degas, put the sample in a vacuum (house vacuum or
even an aspirator will do) for 10 minutes or so. Bubbles will form
on the walls- these should be removed by gentle tapping.
Alternatively, sparge the sample with N2 or Ar for 5 minutes.
2. Include a carrier in sample. There are three surfaces where
fluorescent material may bind- the air-liquid meniscus, the
centerpiece walls and the windows. Of these, the windows are the
most frequent source of trouble. We have tried a number of
treatments- coating with PEG, treatment with chloroethyl silane
and adding a small quantity of detergent to the sample. The best
treatment seems to be to include a small amount (0.1 mg/ml) of
carrier protein. We have had good luck with ovalbumin, serum
albumin and kappa casein. I agree with Dmitry, though, that you
have to be aware that the carrier protein may not be an inert
component. I know of one example where there was a moderately
tight interaction between a labeled protein (gamma subunit of PDE)
and ovalbumin, leading to a "surprise" 4 s peak. Switching to
serum albumin got rid of the problem.
3. Loss of sample to the walls is very dependent on the material. I
have seen examples of proteins that are fine without a carrier at
concentrations 30 pM, and have seen an example (tubulin
dissociation) where no carrier protein seems to prevent loss of
protein (the monomer in this case). Our rule of thumb is to
include 0.1 mg/ml ovalbumin in solvents.
4. Do not despair if the signal level in the sample is less than the
centerpiece. The epoxy used in the centerpieces is fairly
fluorescent, so there may be a dip in the intensity for the sample
when viewed in the "Cell settings" window. That window provides an
estimate of the signal to noise (in the bar above the graph).
However I am not entirely satisfied with the calculation (which
uses the signal and noise in the sample and compares this with the
signal and noise at a position where there is no sample). The S/N
is given in decibels (dB), which is 20*log10(S/N). Making this
calculation better is on my to-do list.
Best wishes to all-
Tom
Borries Demeler wrote:
> Thanks, Dmitry, this is very much in line with what I have observed,
> in the hundreds of nanomolar the signal to noise is usually acceptable,
> below the signal gets too noisy. Regarding the sticking problem you
> might try adding a little unlabeled carrier protein like BSA. In the low
> nanomolar range I have not had much luck with signal. But it is good to
> know that someone else gets a good signal at 1 nM label concentration.
>
> -Borries
>
>
>> Hi Borries,
>> so far I was only able to get down to 250e-9 of labelled proteins.
>> Signal is good, but I have problems with protein sticking to cells and
>> not willing to sediment. But this is not a sensitivity issue.
>> With pure fluorescein, I was able to get reasonable signal from 1 nM.
>> But i do not have any S/N values, unfortunately.
>> regards, Dmitry
>>
>> Borries Demeler wrote:
>>
>>> Hi Dmitry,
>>>
>>> Thanks so much for the information. I am trying to get a feel for the
>>> molarity of label necessary to get a good signal (i.e., at least 50-100
>>> to 1 signal to noise). This should be relatively independent of PMT
>>> etc (increasing the gain will also give you more noise, so I am asking
>>> for what can be seen under the best circumstances). Let's assume our
>>> instrument is properly aligned. What molarity of Alexa 488 label is
>>> necessary to get at least that good a signal? Does anyone on this list
>>> have a ballpark figure? Right now I don't have either dye on hand,
>>> and just want to decide if the proposed experiment is feasable.
>>> Is it 10-8, 10-9, 10-10 M? Just the order of magnitude would be sufficient
>>> at this point.
>>>
>>> Thanks, -Borries
>>>
>>>
>>>> Dear Borries,
>>>> Alexa 488 has properties very similar to fluorescein.
>>>> Its extinction coefficient is about 80,000 vs 65,000 for fluorescein,
>>>> excitation and emission spectra are the same.
>>>> Its apparent strength is improved photo-stability, but it may not as
>>>> obvious because fluorescein works quite well.
>>>> Basically, you can treat it as if it was fluorescein.
>>>>
>>>> On a more general note, fluorescence intensity is a relative signal. It
>>>> will depend on PMT settings, optics alignment, and will vary from
>>>> instrument to instrument.
>>>> I calibrate the signal intensity in XLF by
>>>> 1) having one sector with buffer, to measure background level
>>>> 2) by recording a scan at low speed at the beginning of experiment and
>>>> assuming the intensity reading for the loading concentration
>>>> 3) because optical density of samples is low (<0.06, ie <1 uM), inner
>>>> filter effect could be ignored, and the signal could be assumed to
>>>> directly proportional to concentration of solute.
>>>> best regards, Dmitry
>>>>
>>>>
>>>> Borries Demeler wrote:
>>>>
>>>>> Can someone tell me what the molar emission of Alexa 488 in units of
>>>>> the XLF? Basically, I want to know the molarity of Alexa label necessary
>>>>> to get a clean, relatively noise-free signal in the XLF. I haven't done
>>>>> the experiment, but would like to decide if a planned experiment is feasable
>>>>> without having to go through the calibration for now.
>>>>>
>>>>> Thanks for any pointers.
>>>>>
>>>>> -Borries
>>>>> _______________________________________________
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>>>>>
>>>>>
>>>>>
>>>> --
>>>> Dr. Dmitry Veprintsev
>>>> MRC Centre for Protein Engineering
>>>> Hills Road, Cambridge CB2 2QH, UK
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>>>>
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>>>
>> --
>> Dr. Dmitry Veprintsev
>> MRC Centre for Protein Engineering
>> Hills Road, Cambridge CB2 2QH, UK
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