Re: Strange Photo conversion

Posted by Cameron Nowell-3 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-Photo-conversion-tp7586797p7586798.html

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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
>
>
> --
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> *Cameron J. Nowell*
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