Capturing fluorescent proteins on agarose beads

[Hirani, Nisha]

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Hi everyone,
 
I have a question regarding the capture of fluorescent proteins on agarose
beads.

I¹ve captured four different fluorescent proteins on agarose beads by two
different methods and have obtained different images, but was expecting very
similar results.
 
Method 1 ­ Fluorescent protein was captured on agarose beads using an
antibody (against the fluorescent protein) and results show bright
expression with a Ohalo effect¹ of fluorescence around the agarose beads.
 
Method 2 ­ Fluorescent proteins containing the S-tag sequence (at the C
terminal of protein) were captured on agarose beads coated with S-protein
(S-tag binds to S-protein) and the images obtained were not the Ohalo
effect¹ as seen previously but the bead outline and fluorescence expression
appear fuzzy.
 
Has anyone come across this before or have any ideas as to what could be
causing the differences seen?
 
Any help would be greatly appreciated.
 
Nisha

[Rickman, Colin]

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Hi

This will be dependent on the porosity of the two types of beads you are
using. How were the S-tag protein and antibodies coupled to the agarose
beads (e.g.Protein A, CNBr etc..)? If the affinity agent is only on the
surface of the bead you would expect to see a halo. If the fluorescent
protein has been immobilised throughout the structure of the bead then
it would look "fuzzy" - dependant on the type of microscope youa re using.

Contact me off list if you want more information.

Regards

Colin

Dr Colin Rickman
Life Science Interface
Department of Chemistry (WP2.03)
School of Engineering and Physical Sciences
Heriot-Watt University
Edinburgh
EH14 4AS

Tel: [phone] (WP2.03 - Office)
Tel: [phone] (WP3.17 - Laboratory)

http://www.lifescienceinterface.hw.ac.uk
http://www.eps.hw.ac.uk/departments/chemistry/cr.htm

On 14/06/2011 13:50, Hirani, Nisha wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
>
> I have a question regarding the capture of fluorescent proteins on agarose
> beads.
>
> I¹ve captured four different fluorescent proteins on agarose beads by two
> different methods and have obtained different images, but was expecting very
> similar results.
>
> Method 1 ­ Fluorescent protein was captured on agarose beads using an
> antibody (against the fluorescent protein) and results show bright
> expression with a Ohalo effect¹ of fluorescence around the agarose beads.
>
> Method 2 ­ Fluorescent proteins containing the S-tag sequence (at the C
> terminal of protein) were captured on agarose beads coated with S-protein
> (S-tag binds to S-protein) and the images obtained were not the Ohalo
> effect¹ as seen previously but the bead outline and fluorescence expression
> appear fuzzy.
>
> Has anyone come across this before or have any ideas as to what could be
> causing the differences seen?
>
> Any help would be greatly appreciated.
>
> Nisha

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