The Biofilm Exopolysaccharide Polysaccharide Intercellular Adhesin—A Molecular and Biochemical Approach - Bacterial Pathogenesis: Methods and Protocols - page 96

3. Resuspend cell pellet in 20 μL 0.5 M EDTA, pH 8.0 (final volume: 1/50 of growth
culture volume).
4. Extract surface-located PIA by boiling cells for 5 min at 100 °C in a 2-mL
(safelock) microtube.
5. Centrifuge samples at 10,000 × g for 5 min at 4 °C.
6. Transfer clear supernatant to a new microtube.
7. Freeze the extract at -20 °C for months ( see Note 2 ).

3.2.1.2. Isolation of PIA from the Culture Filtrate
1. Harvest 1 mL of a 16-h staphylococcal culture at 10,000 × g for 5 min at 4 °C.
2. Transfer clear supernatant into a new microtube.
3. Concentrate supernatant with an ultrafiltration device (Amicon Ultrafree-MC,
YM-10) by centrifugation at 4000 × g for 30 min at 4 °C.
4. Repeat step 3 until the final concentration ratio is about 50-fold ( see Note 3 ).
5. Freeze the extract can be frozen at -20 °C.

3.2.2. Highly Pure PIA for Immunization and Chemical Analysis
Highly pure PIA is required to raise specific antibodies against PIA and for
chemical analysis of PIA deacetylation. For standard calibration, high-quality
PIA is preferable.

3.2.2.1. Isolation of Cell-Surface-Attached Highly Pure PIA
1. Harvest1Lofa16-h staphylococcal culture by centrifugation at 3000 × g for
15 min at 4 °C.
2. Wash bacterial cell pellet with 500 mL PBS buffer and repeat centrifugation
step.
3. Resuspend bacterial cell pellet in 20 mL 0.5 M EDTA, pH 8.0 (final volume:
1/50 of growth culture volume).
4. Extract surface-associated PIA by boiling cells for 5 min at 100 °C.
5. Centrifuge sample at 3000 × g for 30 min at 4 °C and transfer clear supernatant
to a dialysis membrane ( 10 kDa cut off).

6. Dialyze PIA-containing extracts against distilled water for 2 × 12 h at 4 °C
and subsequently digest with DNase (0.5 mg/mL final concentration), RNase
(0.5 mg/mL final concentration), lysostaphin (0.5 mg/mL final concentration),
and lysozyme (0.5 mg/mL final concentration) at 37 °C for 16 h, followed by
incubation with proteinase K (4 mg/mL final concentration) at 37 °C for 16 h.
7. Centrifuge sample at 28,000 × g at 4 °C for 30 min.
8. Concentrate clarified supernatant about fivefold with Amicon Centriprep YM-10
centrifugal concentrators.
9. Performsize exclusion chromatography (SEC) (AktaChromatography system, GE
Healthcare Life Sciences, Piscataway, NJ) of PIA in maximally 10-mL aliquots
on a HiLoad 26/60 Superdex 200 gel filtration column. Use 20 m M sodium phos-
phate (pH 7.0) and 150 m M sodium chloride as a buffer at a flow rate of 3 mL/min.

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