Detergent compatability in T cell assays?

[Guy Hermans]

In article
<Pine.OSF.3.96.971027112503.9217A-100000 at saul5.u.washington.edu>, Thomas
Arroll <twa at u.washington.edu> wrote:

> Hi,
> 
> I am working on a project which involves looking at T cell responses to
> recombinant bacterial proteins.  One problem I have been having is that
> some of the recombinant proteins that I have been expressing and affinity
> purifying totally crash out of solution when I dialize out the denaturant
> (8 M urea).  I have tried a number of standard tricks to try to keep these
> proteins in solution with no avail including; 1) Step dialysis into an
> intermediate urea concentration solution, 2) Dialysis into 10 mM Tris (pH
> 8.0). 3)  Dilution of protein prior to dialysis.  So, in regards to this
> problem I have a few questions; 1)  Does anyone have any ideas of
> additional things to try in order to keep my proteins in solution?, 2)
> Are there any detergents that I can use that might be compatible with T
> cell proliferation assay (mixed lymphocyte assay using rabbit
> splenocytes).  3)  Are T cell responses to insoluble particulate antigens
> comparable to that of soluble antigens.  As per my last question I have
> read that macrophages will take up, process and present particulate
> antigens more efficiently than soluble antigens, is this generally true?   
> 

Try dissolving the proteins in pure ethanol (pro analysi quality; filter
sterile after dissolving the protein). Sometimes smaller quantities (say,
10%) will to.

Second solution (pun intended) is using a low (0,01%) solution of Tween-20.

Another option would be to use a 10%DMSO solution - but don't try this one
untill all else fails. DMSO is quite toxic. 

It's absolutely imperative to filter the solution after dissolving the
solution. Not only for sterility reasons: microparticles might still be
present and mess up protein determination assays afterwards, or may
influence the behaviour of the cells.

Good luck,

Guy

--------------------------------------------
Guy Hermans, PhD student
MS research Unit             Tel 0032 (0) 11/26.92.07
Dr. L. Willems-Institute     Fax 0032 (0) 11/26.92.09
University Campus            E-mail ghermans at luc.ac.be
B-3590 Diepenbeek
Belgium
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