Protein immobilization on coverslips

[F Javier Diez Guerra]

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Hello,

We want to assay in vitro the FRET efficiency of several CFP-YFP
biosensors by immobilizing them on coverslips.
Can anybody advice on protocols for the coverslip immobilization step?

Thanks
--

Fco. Javier Díez Guerra, PhD
Profesor Titular
Centro de Biología Molecular Severo Ochoa
C/ Nicolás Cabrera, 1
Universidad Autónoma
Ctra Colmenar Viejo Km 15
Cantoblanco, 28049 Madrid
SPAIN

phone:  [phone]
e-mail: [hidden email]

[daj1u06]

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I guess that any of the standard protocols for preparing home-brew ELISA
plates should work. We also recently came across a very simple method for
sticking cells down using alcian blue - referenced in the following which
might also have other suggestions worth trying:

http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291097-0320%2820000401%2939:4%3C295::AID-CYTO7%3E3.0.CO;2-X/full

Alcian blue seems to work very well for sticking down primary mast cells for
confocal imaging - exactly what it is interacting with on the cell surface
I'm not sure - might depend on whether your proteins are glycosylated.

[Stabley, Daniel]

Javier,

You could give this type of linkage a shot:

http://www.nature.com/nmeth/journal/v9/n1/full/nmeth.1747.html

Daniel Stabley
Postdoctoral Research Fellow
Developmental Neurobiology
St. Jude Children's Research Hospital

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Fco. Javier Díez Guerra
Sent: Monday, January 18, 2016 9:58 AM
To: [hidden email]
Subject: Protein immobilization on coverslips

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Hello,

We want to assay in vitro the FRET efficiency of several CFP-YFP biosensors by immobilizing them on coverslips.
Can anybody advice on protocols for the coverslip immobilization step?

Thanks
--

Fco. Javier Díez Guerra, PhD
Profesor Titular
Centro de Biología Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Universidad Autónoma Ctra Colmenar Viejo Km 15 Cantoblanco, 28049 Madrid SPAIN

phone:  [phone]
e-mail: [hidden email]

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