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Hi All,

Yep it was self quenching. Once we took the dilution down it stopped
getting brighter. Actually got it down dilute enough to see textbook step
wise bleaching of single fluors :)

Cheers

Cam

On 6 May 2017 at 02:22, Kilgore, Jason A. <[log in to unmask]>
wrote:

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>
> Hi, Cameron,
>
> I don't know for certain, but I wonder if what you are seeing is a dye-dye
> quenching effect.
>
> In other words, with some dyes (I'm not certain about Cy5, though), if
> over-labeled, will have FRET-based dye-dye quenching.  If you then
> photobleach the sample, it reduces the quenching, leading initially to a
> brighter signal.  (If the photobleaching continues, the signal will plateau
> at the point when completely unquenched, then will decrease as the dye
> molecules continue photobleaching).
>
> If this is what is happening, then as you supposed, reducing the dye
> concentration should reduce the dye-dye quenching.
>
> Cheers,
>
> Jason
>
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Cameron Nowell
> Sent: Thursday, May 04, 2017 8:55 PM
> To: [log in to unmask]
> Subject: Re: Strange Photo conversion
>
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>
> The final experiment is to couple O6-benzyl-guanine via succinamide ester
> to then allow attachment of a protien via a genetically encoded SNAP tag.
>
> The Cy5 and poly d was just a proof of concept to see how stable the
> labelling would be as  we tried the other way and couldn't see any signals.
>
>
>
> On 5 May 2017 at 12:09, <[log in to unmask]> wrote:
>
> > Sorry I still don't understand. Do you plan to label protein with Cy5
> > separately first and then stick it to the polylysine in the channel
> > after purification? Right now it sounds like you are just covalently
> > attaching dye to lysine in the channel and I don't get why you would
> > want that. Yes, lower concentrations of dye can reduce quenching if it
> > is quenching, though as I said before, I am not certain.
> >
> > On May 4, 2017, at 6:32 PM, Cameron Nowell <[log in to unmask]>
> > wrote:
> >
> > The plan is to use the poly d to anchor our cy5 that will be coupled
> > to a protein. Then come in with a fluorescent ligand in a different
> > channel and look at binding.
> >
> > So if it's a quenching type thing would lowering the concentration of
> > dye help?
> >
> >
> > On 5 May 2017 11:27 am, <[log in to unmask]> wrote:
> >
> > Are you trying to label the polylysine? In principle, heavily labeled
> > stuff can start out quenched and then with illumination to photobleach
> > a few molecules the system gets brighter. I've seen this a few times
> > in the past, though I have no way to know if you are in this strongly
> > quenched regime.
> >
> > > On May 4, 2017, at 6:05 PM, Cameron Nowell
> > > <[log in to unmask]>
> > wrote:
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> > posting.
> > > *****
> > >
> > > Hi List,
> > >
> > > We are trying to get some experiments up and running and have hit a
> > bizarre
> > > artifact with Cy5 getting photo converted and becoming very very
> bright.
> > >
> > > The experimental desing is as follows
> > > - fluorodish or ibidi channel slide
> > > - coated with Poly-D-Lysine in PBS
> > > - Washed PBS
> > > - Washed Bi-carbonate pH 8.6
> > > - Couple Cy 5 succinamide ester bi-carb pH 8.6
> > > - Wash Bi-carb 8.6
> > > - Wash PBS
> > >
> > > IMaging on either a TIRF or confocal system we get a rapid and
> > > robust increase in fluorescene in the Cy5 channel. Exited at any
> > > wavelegth (405,
> > > 488 or 647).
> > >
> > > Any ideas on what could be going on?
> > >
> > > Cheers
> > >
> > > Cam
> > >
> > >
> > > --
> > >
> > > *Cameron J. Nowell*
> > >
> > > Head
> > >
> > >
> > >
> > > Imaging, FACS and Analysis Core
> > >
> > > Monash Institute of Pharmaceutical Sciences
> > >
> > > Monash University
> > >
> > > 399 Royal Parade
> > >
> > > Parkville, VIC, 3052
> > >
> > > Australia
> > >
> > >
> > >
> > > *Email:* [log in to unmask]
> > >
> > > *Phone: *+61 422882700
> > >
> > >
> > >
> > > *LinkedIn: *Profile <http://au.linkedin.com/pub/ca
> > meron-nowell/23/57/884/>
> > >
> > > *Research Gate: * Profile
> > > <http://www.researchgate.net/profile/Cameron_Nowell>
> > >
> > > *Google Scholar:* Profile
> > > <https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>
> > >
> > > *PubMed Bibliography: *Profile
> > > <http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibl
> > iography/47922177/public/?sort=date&direction=ascending>
> >
> >
> >
>
>
> --
>
> *Cameron J. Nowell*
>
> Head
>
>
>
> Imaging, FACS and Analysis Core
>
> Monash Institute of Pharmaceutical Sciences
>
> Monash University
>
> 399 Royal Parade
>
> Parkville, VIC, 3052
>
> Australia
>
>
>
> *Email:* [log in to unmask]
>
> *Phone: *+61 422882700
>
>
>
> *LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>
>
> *Research Gate: * Profile
> <http://www.researchgate.net/profile/Cameron_Nowell>
>
> *Google Scholar:* Profile
> <https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>
>
> *PubMed Bibliography: *Profile
> <http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/
> bibliography/47922177/public/?sort=date&direction=ascending>
>



-- 

*Cameron J. Nowell*

Head



Imaging, FACS and Analysis Core

Monash Institute of Pharmaceutical Sciences

Monash University

399 Royal Parade

Parkville, VIC, 3052

Australia



*Email:* [log in to unmask]

*Phone: *+61 422882700



*LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>

*Research Gate: * Profile
<http://www.researchgate.net/profile/Cameron_Nowell>

*Google Scholar:* Profile
<https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>

*PubMed Bibliography: *Profile
<http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibliography/47922177/public/?sort=date&direction=ascending>