GFP bleaching: folding problem?

[offterdi1]

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Dear all,

A colleague of mine experienced a special bleaching problem.

A specific fusion construct of EGFP showed very rapid bleaching compared to
unfused EGFP. I know that the overall brightness of fusions is often
significant lower than of unfused EGFP (which was also true inher case) but
I was not aware of such a
bleach-specific difference. Can these be explained by unsufficient/partial
folding of the EGFP within the fusion. In that case I d recommend to her to
use superfolderGFP instead but I just wonder whether that explanation makes
any sense to you?

Many thanx!

***********************
Martin Offterdinger, PhD
Medical University Innsbruck
Biocenter
Division of Neurobiochemistry
Biooptics
Fritz-Pregl-Strasse 3
A-6010 Innsbruck
Austria
***********************
http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Main.html

[Guy Cox-2]

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I'm no expert in this field but I think any meaningful answer would
require knowledge of what the construct was.

As an extreme example, if you conjugated eGFP to a peroxidase you could
probably expect this.

                                      Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Martin Offterdinger
Sent: Thursday, 29 September 2011 7:08 PM
To: [hidden email]
Subject: GFP bleaching: folding problem?

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Dear all,

A colleague of mine experienced a special bleaching problem.

A specific fusion construct of EGFP showed very rapid bleaching compared
to
unfused EGFP. I know that the overall brightness of fusions is often
significant lower than of unfused EGFP (which was also true inher case)
but
I was not aware of such a
bleach-specific difference. Can these be explained by
unsufficient/partial
folding of the EGFP within the fusion. In that case I d recommend to her
to
use superfolderGFP instead but I just wonder whether that explanation
makes
any sense to you?

Many thanx!

***********************
Martin Offterdinger, PhD
Medical University Innsbruck
Biocenter
Division of Neurobiochemistry
Biooptics
Fritz-Pregl-Strasse 3
A-6010 Innsbruck
Austria
***********************
http://www.i-med.ac.at/neurobiochemistry/neurobiochemistry/Biooptics/Mai
n.html

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[offterdi1]

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The construct was not targeted to any organelle or so I don't know the
details but it was not something very unususal at the first glimpse.
martin