GFP bleaching

[M. Jarnik]

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

[Alberto Diaspro]

Temperature increase makes photobleaching faster..
Il giorno 10/nov/08, alle ore 15:36, M. Jarnik ha scritto:

> 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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Department of Physics, University of Genoa,
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[Xuejun Sun]

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: [phone]
Fax:     [phone]

-----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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[Andreas V]

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: [phone]
> Fax:     [phone]
>
> -----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
>
>
> This e-mail and any attachments may contain confidential and
> privileged information. If you are not the intended recipient,
> please notify the sender immediately by return e-mail, delete this
> e-mail and destroy any copies. Any dissemination or use of this
> information by a person other than the intended recipient is
> unauthorized and may be illegal.