http://confocal-microscopy-list.275.s1.nabble.com/Strange-Photo-conversion-tp7586797p7586799.html
> 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
> >
> >
> >
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>
*Cameron J. Nowell*