Posted by
Andreas V on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-bleaching-tp1480738p1482046.html
Dear All,
I was wondering whether or not somebody would like to share his opinion on
the following "GFP-FRAP issue". Have been discussing this lately with a
colleague of mine in the lab:
Since EGFP itself has a certain maturation time (t1/2 = 0.3h - depending on
various conditions), this would apparently cause difficulties/problems in
FRAP experiments for proteins with a seemingly slow turnover - taking into
consideration that newly translated proteins will need certain time before
starting to "emit".
Is there anybody out there who has struggled with the same concern and can
offer a solution/answer to it?!
We came up with the idea to bleach a rather huge field and see if the
recovering is occurring first at the borders of the bleached area (sort of
FLIP/FRAP combination).
Best regard,
Andreas
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]] On Behalf Of Xuejun Sun
> Sent: Montag, 10. November 2008 20:17
> To:
[hidden email]
> Subject: Re: GFP bleaching
>
> Your time resolution is maybe too low - either recovering happened or
> sample
> moved... Here are few options: zoom up/increase laser power and reduce
> number of bleaching cycles, reduce image area and/or use higher frame
> rate.
>
> Good luck!
>
> Xuejun
>
> Xue-jun Sun, Ph.D.
> Dept. Exp. Oncology
> Cross Cancer Institute
> 11560 University Ave,
> Edmonton Alberta T6G 1Z2
> Canada
> Phone: (780) 432-8898
> Fax: (780) 432-8425
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]] On
> Behalf Of M. Jarnik
> Sent: Monday, November 10, 2008 7:36 AM
> To:
[hidden email]
> Subject: GFP bleaching
>
> Hi Listers,
>
> I am trying some FRAP experiments on our Nikon C1 confocal. In
> particular, I
> would neet to
> bleach GFP on microtubules in mitotic cells. While bleaching in mounted
> fixed samples
> seems to work well, we have problems in life spls (using a heated
> chamber).
> Any magic
> touch we might be missing?
>
> Thanks,
>
> Michal
>
>
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