PFA fixing + triton, what happens to surface proteins ?

[Jean-Pierre CLAMME-2]

Hi,

It might be a trivial question, but I was just wondering, if I fix cells  with PFA and thus cross-link surface proteins or other molecules on the surface of the cell, what would happen to them if I permeabilize the cell using 0.1% Triton? Will  they disappear with the membrane ? Will they stay cross-linked to some transmembranar proteins and thus with the inside of the cell ?

Any comments are welcome.

Thanks,

JP

[Boswell, Carl A - (cboswell)]

It is my understanding that detergents, when in the proper concentration, do not dissolve membranes as much as open holes so that hydrophilic molecules can move freely between inside and outside the cell.  In other words, not all the lipid is lost.   

 In addition, the cross-linking of proteins can make large complexes that become insoluble.  The best answer is to try it.   Fixing and staining surface proteins is a routine practice so loss of surface antigens would not seem to be a common problem.

 

My experience has been that antibodies can make it inside fixed cells without any detergent processing.  Because of that, I've had to treat live cells with primary antibody to ensure that no internal staining of the same antigen would occur.

 

Carl

 

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

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Sent: Thursday, March 12, 2009 2:28 PM

Subject: PFA fixing + triton, what happens to surface proteins ?

Hi,

It might be a trivial question, but I was just wondering, if I fix cells  with PFA and thus cross-link surface proteins or other molecules on the surface of the cell, what would happen to them if I permeabilize the cell using 0.1% Triton? Will  they disappear with the membrane ? Will they stay cross-linked to some transmembranar proteins and thus with the inside of the cell ?

Any comments are welcome.

Thanks,

JP