[FDS] Alexa 488

[Michael Frederick Bailey]

Hi Borries,

I have managed to get reasonable data for a 68 kDa Alexa488 labeled
protein at a probe concentration of 5 nM.  I can send the data if you
are interested.

Cheers,

Michael Bailey

-----Original Message-----
From: fds-bounces at biochem.uthscsa.edu
[mailto:fds-bounces at biochem.uthscsa.edu] On Behalf Of Borries Demeler
Sent: Sunday, 3 December 2006 5:33 AM
To: fds at biochem.uthscsa.edu
Subject: Re: [FDS] Alexa 488

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
> >>> _______________________________________________
> >>> FDS mailing list
> >>> FDS at biochem.uthscsa.edu
> >>> http://biochem.uthscsa.edu/mailman/listinfo/fds
> >>>
> >>>
> >> -- 
> >> Dr. Dmitry Veprintsev
> >> MRC Centre for Protein Engineering
> >> Hills Road, Cambridge CB2 2QH, UK
> >> _______________________________________________
> >> FDS mailing list
> >> FDS at biochem.uthscsa.edu
> >> http://biochem.uthscsa.edu/mailman/listinfo/fds
> >>
> > 
> > _______________________________________________
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> > 
> > 
> 
> -- 
> Dr. Dmitry Veprintsev
> MRC Centre for Protein Engineering
> Hills Road, Cambridge CB2 2QH, UK
> _______________________________________________
> FDS mailing list
> FDS at biochem.uthscsa.edu
> http://biochem.uthscsa.edu/mailman/listinfo/fds
> 

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