FCS of proteins in solution

[els vanstreels]

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Dear all,

We are trying to perform fluorescence correlation spectroscopy measurements on proteins in solution (PBS buffer).  Our sample is on a standard n0. 1.5 coverglass placed on an inverted microscope.  We are experiencing a lot of problems with adherence of the proteins to the glass surface, in a sense that the signal is decreasing rapidly after bringing the sample onto the glass.  Is there a way to circumvent this problem, eg by applying some coating to the coverglass or performing the measurements in an other type of buffer?

Thanks for all suggestions,

 

Els

 

 

Dr. Ir. Els Vanstreels

Rega Institue for Medical Research

Minderbroedersstraat 10

3000 Leuven

Belgium

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[mmodel]

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Can you add a large excess of non-fluorescent protein, such as BSA, to your solution to block the glass?

 

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of els vanstreels
Sent: Thursday, September 27, 2007 9:10 AM
To: [hidden email]
Subject: FCS of proteins in solution

 

Dear all,

We are trying to perform fluorescence correlation spectroscopy measurements on proteins in solution (PBS buffer).  Our sample is on a standard n0. 1.5 coverglass placed on an inverted microscope.  We are experiencing a lot of problems with adherence of the proteins to the glass surface, in a sense that the signal is decreasing rapidly after bringing the sample onto the glass.  Is there a way to circumvent this problem, eg by applying some coating to the coverglass or performing the measurements in an other type of buffer?

Thanks for all suggestions,

 

Els

 

 

Dr. Ir. Els Vanstreels

Rega Institue for Medical Research

Minderbroedersstraat 10

3000 Leuven

Belgium

Tel.: [phone]

Fax.: [phone]

 

Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information.

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Els-
There is a protein in plasma (also in serum) that binds tightly to
glass.  The replacement series of proteins was worked out years
ago by Leo Vroman of the New York City VA Hospital, if you wish to
look up the data.

If you put a 10% solution of plasma or serum on the surface, it will
coat it completely and prevent or at least drastically limit the
adsorption of your fluorescent proteins.  See anybody who does
tissue culture, and they will have the supplies you need.
Carol

[Csucs Gabor]

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Dear Els,

Proteins do adsorb everywhere, pretty much independent on the buffer you
are using (of course the amount can be varying). On way to avoid this
that you start your experiment with a high concentration of proteins -
let the saturate the surface and start you FCS measurements afterwards.
Actually if you use a large enough cuvette/sample volume and you measure
the FCS signal as far from the glass as it is possible - the this should
solve your problems. Another way to deal with your problem avoid this
(as someone mentioned it before) is the passivation of the surface. This
can be achieved either by incubating your glass slide in serum albumin -
this is the simplest way (although not particularly stable), but you can
use other , dedicated materials like Pluronics or PLL-g-PEG (we have
published couple of papers with this latter). But there are of course
many other materials which one could use. Surface passivation is a
rather"standard" biomedical/biotechnological problem so there are many
papers existing on the subject.

Cheers    Gabor

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
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e-mail: [hidden email]