http://confocal-microscopy-list.275.s1.nabble.com/Strange-Photo-conversion-tp7586797p7586808.html
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
On 6 May 2017 at 02:22, Kilgore, Jason A. <
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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
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]]
> On Behalf Of Cameron Nowell
> Sent: Thursday, May 04, 2017 8:55 PM
> To:
[hidden email]
> 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, <
[hidden email]> 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 <
[hidden email]>
> > 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, <
[hidden email]> 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
> > > <
[hidden email]>
> > wrote:
> > >
> > > *****
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> > >
> > > 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
> > >
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> >
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>
> --
>
> *Cameron J. Nowell*
>
> Head
>
>
>
> Imaging, FACS and Analysis Core
>
> Monash Institute of Pharmaceutical Sciences
>
> Monash University
>
> 399 Royal Parade
>
> Parkville, VIC, 3052
>
> Australia
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> *Email:*
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> *PubMed Bibliography: *Profile
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