Posted by
Kilgore, Jason A. on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-Photo-conversion-tp7586797p7586801.html
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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:
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > Post images on
http://www.imgur.com and include the link in your
> 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:*
[hidden email]
> >
> > *Phone: *+61 422882700
> >
> >
> >
> > *LinkedIn: *Profile <
http://au.linkedin.com/pub/ca> meron-nowell/23/57/884/>
> >
> > *Research Gate: * Profile
> > <
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> >
> > *Google Scholar:* Profile
> > <
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> >
> > *PubMed Bibliography: *Profile
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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
*Email:*
[hidden email]
*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>