http://confocal-microscopy-list.275.s1.nabble.com/Is-DMSO-photobleaching-autofluorescent-proteins-tp7579990p7579996.html
happens to be plant cells but principle is general) against solvents.
We found biological effects of 1% DMSO (and of 0.1% ethanol). For
1% DMSO (or 0.1% ethanol). I would not be surprised if animal/fungal
cells also were affected by 1% DMSO. Over the years we have used a
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>Dear Josef,
>
>indeed, it is possible that DMSO quenches the fluorescence FPs:
>
>
http://pubs.acs.org/doi/abs/10.1021/j100372a091>
http://www.ncbi.nlm.nih.gov/pubmed/2457301>
>If it is the case, the effect ought to be reversible (immediately); if
>the protein moiety is dentured, I guess it should be irreversible.
>
>Hope this helps,
>Regards,
>Jurek Dobrucki
>--
>Jerzy Dobrucki, Ph.D., D.Sc.
>Professor of Biophysics
>Head, Division of Cell Biophysics
>Faculty of Biochemistry, Biophysics and
> Biotechnology
>Jagiellonian University
>ul. Gronostajowa 7
>30-387 Krakow, Poland
>tel. +48 12 664 6382; fax. +48 12 664 5503
>
[hidden email]
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http://www.wbbib.uj.edu.pl/helios>
>
>
>
>On Thu, Mar 14, 2013 at 8:01 PM, Kelly Lundsten
><
[hidden email]> wrote:
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>> Hi Josef,
>>
>> It's much more likely that you are denaturing the fluorescent
>>protein. Simple synthetic organic fluors like Alexa Fluors or Cy
>>dyes, are often reconsistituted entirely in DMSO in their reactive
>>form, for that purpose to protect them from oxygen. DMSO does not
>>hurt the actual fluorescent molecule itself. I would absolutely
>>imagine it can hurt protein, though. With fluorescent proteins,
>>the chromophore at the center of the beta barrel of a GFP requires
>>the protein component to remain rigid. Should the protein
>>component relax or denature, the chromophore at the center of the
>>molecule is no longer rigid itself and cannot resonate the energy
>>it absorbs efficiently (quantum efficiency). The same effect is
>>seen with methanol or other denaturing solvents and fluorescent
>>proteins. The effect is not photobleaching, just a loss of quantum
>>efficiency.
>>
>> Your cells also can't be so happy with the presence of the DMSO.
>>What most people would do in this instance is ask themselves 1. Can
>>the drug be dissolved in something besides DMSO and 2. If not, can
>>you concentrate the drug more so as to deliver the least amount of
>>DMSO as is possible.
>>
>> Good luck,
>> Kelly
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>>[mailto:
[hidden email]] On Behalf Of Josef
>>Gotzmann
>> Sent: Thursday, March 14, 2013 9:42 AM
>> To:
[hidden email]
>> Subject: Is DMSO photobleaching autofluorescent proteins?
>>
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>>
>> Dear List,
>>
>> I have a technical questions:
>> When we live image cells (in buffered saline, pH7.4) expressing GFP- and
>> mCherry-labeled proteins, respectively, on our confocal and we add a drug
>> (dissolved in DMSO) we rapidly (within seconds) lose fluorescence of both
>> dyes. After a very sharp drop down to 50% of intensity within the first 2
>> minutes, we reach a plateau (2-5minutes), followed by another less sharp,
>> though steady, decrease in fluorescence for another 2 minutes.
>> This is the effect we see with an end concentration of 1% DMSO, which is
>> completely absent when are scaling down the DMSO concentration to 0,1% !
>> We have already ruled out that it's simple bleaching due to lightning
> > conditions.
>> My understanding was that the OH-radical scavenging properties of DMSO
>> should more stabilize fluorescence!?
>> Anyone an idea how DMSO can act like that?
>>
>> thx
>> Josef
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