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In-Depth Information
5. A similar method has been employed to purify poly-
N
-acetylglucosamine (PNAG
from
Staphylococcus aureus
strain MN8
(8)
. Production of PNAG and PIA is
encoded by the same gene locus in staphylococci, the
icaADBC
operon. Both
polysaccharides are considered to be the same. PNAG-containing samples were
fractionated by SEC with a Sephacryl S-300 column and 0.1
N
HCl/0.15
M
NaCl
buffer
(8,9)
.
6. Incubation with the primary PIA antiserum up to 16 h increases the strength of
the immunological reaction.
7. These methods do not require further purification of PIA before chromatography.
However, purging the columns extensively after several runs is necessary, as
particularly the PolarPac Wax column tends to bind negatively charged polymers
(such as teichoic acids) that need to be removed because they interfere with the
repulsion mechanism of positively charged PIA on this column, which is needed
for optimal purification of deacetylated PIA
(4)
.
8. This assay is a modified ninhydrin test to determine the degree of deacetylation
in PIA. The reagent TNBS has been shown to react specifically with free amino
groups to give trinitrophenyl (TNP) derivatives that can be measured with a UV
spectrophotometer at 335 nm
(10)
. Chemically, complete deacetylation of PIA
can be achieved by treatment with strong acid or base for several hours.
Acknowledgments
This work was supported by the Intramural Research Program of the National
Institutes of Allergy and Infectious Diseases, National Institutes of Health.
References
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