Removing protein from polysaccharide

[Yulia Shifrin]

This is what worked for us:

Preparation of total cellular polysaccharide.

Bacteria were grown overnight in 50 ml LB without NaCl. The OD of the
cultures was adjusted to 1.0, and the cultures were centrifuged and
resuspended in 500 μl of phosphate-buffered saline. An equal volume of
saturated phenol (pH 8.0) was added, and the mixture was incubated for 30
min at 70°C with occasional mixing followed by centrifugation (1 h, 10,000 ×
*g*). The top, aqueous phase was recovered, 2 volumes of 100% ethanol was
added to each sample, and the polysaccharides were allowed to precipitate
for 1 h at −70°C. The samples were centrifuged at 12,000 × *g* for 30 min,
and the pellets were washed with 500 μl of 70% ethanol, recentrifuged, and
lyophilized.

Separation of capsule polysaccharide from LPS.

 The lyophilized total polysaccharide preparations were resuspended in 500
μl of water. The resuspended LPS, which formed micelles, was precipitated by
ultracentrifugation (1 h, 86,000 × *g*). The supernatant containing the
capsule polysaccharide was recovered, and residual LPS contamination was
removed by phase partition with Triton X-114 as described previously.
Briefly, Triton X-114 was added to the supernatant to a final concentration
of 1%. The mixture was incubated at 4°C for 1 h with constant stirring to
ensure a homogenous solution. The mixture was then transferred to a 37°C
water bath, incubated for 10 min, and centrifuged (1 h, 1,000 × *g*) at
25°C. The resulting aqueous phase containing purified capsule polysaccharide
was carefully aspirated
You can look in our paper for the details:
Peleg et al, J Bacteriol. 2005 Aug;187(15):5259-66.

By the way, before you run your capsular polysaccharide on PAGE, check that
it is indeed charged and not too large to enter the gel in the first place.
Best regards

On 11/30/06, Minh Tran Ngoc <minhtn At oucru.org> wrote:
>
> Hello,
>
> Have you known how to remove protein from polysaccharide yet? Now I have
> the same problem. I need the purified polysaccharide of the bateria
> capsule for SDS_PAGE.
>
> Please help me!
>
> Thanks a lot
>
>
>
> Minh
>
>
>
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