LPS purification

[Gerald Pier]

First you need to know several things about your LPS.  Is it from a smooth
organism producing long O side chains or a rough strain lacking O side
chains?  For the former the most routinely used protocol is the
phenol-water extraction procedure of Westphal, Methods Carbohydr. Chem.,
vol 5, 1965 entitled "Bacterial lipopolysaccharides: extraction with
phenol-water and further applications of the procedure."  pages 83-91.  A
rough LPS is often better extracted with phenol-chloroform-petroleum
ether: Galanos, C. Luderitz, O. Westphal, O.  A new method for extraction
of R lipopolysaccharides, Eur. J Biochem vol 9, pg 245-249, 1969.  A third
method involves lysis of the cells with proteinase K and SDS: Hitchcock
and Brown, 1983, Morphological heterogeneity among Salmonella
lipopolysaccharide chemotypes in silver-stained polyacrylamide gels, J.
Bacteriol, vol 154, pgs 269-277.  Finally, we have found that if you lyse
the cells in 1% sarcosyl (sodium lauryl sacrosene) in 0.1 M Tris, pH 7-8
you can release LPS. This last procedure works well with bacteria grown on
agar plates and you can usually get prety good yields from lawns of
bacteria from 10-20 plates. 

After extraction you may want to purify the LPS further.  First
precipitate it by adding ethanol (95%) to a final concentraion of about
80% (you can also use acetone here).  Collect the precipitate, redisolve
in TRIS or PBS buffers around neutral pH, digest the materials with DNAse
and RNase (usually about 0.01 mg/ml for 4 hours at 37 C is fine, you may
want a bit of CaCl2 and MgCl2 in there as well, around 0.001 M is fine)
then add a protease such as Pronase or Proteinase K for about 2 hours. 
Finally, collect the LPS as a precipitate in the ultracentrifuge by
pelleting it at 100,000 X g (usually about 30,000-35,000 rpm), resuspend
it in water and repeat the ultracentrifugation.  You can then lyophilize
the material if you want to weigh it out.

Hope this helps.  Any questions or problems e mail me back.

Jerry Pier
In article <9506192110.AA19654 at umailsrv1.UMD.EDU>,
Lynne_A_WHITEHEAD at UMAIL.UMD.EDU (lw75) wrote:

> Hi there!
> 
> I am interested in protocols for the isolation of LPS from gram neg.
> organisms (I eventually want to use it to raise antibodies against it).  I
> have not yet done a literature search for a protocol because I thought maybe
> there was a common one that people use and could give me a reference.
> I'd appreciate any help or info. you may have!
> 
> Thanks!
> 
> Lynne Whitehead
> U. of Maryland-Micro. Dept.