The Biofilm Exopolysaccharide Polysaccharide Intercellular Adhesin—A Molecular and Biochemical Approach - Bacterial Pathogenesis: Methods and Protocols - page 97

10. Perform immuno-dot blot analysis (described in Subheading 3.3.2. )of
chromatography fractions to determine PIA-containing samples. Most PIA elutes
shortly after the exclusion volume.
11. Dialyze PIA-containing fractions against water for 2 × 12 h (10 kDa cut off)
at 4 °C.
12. Lyophilize, then dissolve PIA in concentrated hydrochloric acid, neutralize with
sodium hydroxide, and buffer the samples with 100 m M sodium phosphate
buffer (pH 7.0).
13. Use highly pure PIA for immunization or for further experiments ( see Note 4 ).
3.2.2.2. Isolation of Highly Pure PIA from the Culture Filtrate
1. Centrifuge5Lofa24-h staphylococcal culture by centrifugation at 3000 × g at
4 °C for 15 min.
2. Sterilize bacterial supernatant by filtration using filtration units (0.22 μm).
3. Concentrate bacterial supernatant to a final volume of 1/20 of the initial volume by
tangential flow filtration using a regenerated cellulose Prep/Scale Spiral Wound
TFF-6 cartridge, a Prep/Scale Holder, and a peristaltic pump at a flow rate of
8 mL/min.
4. Follow steps 6-13 in Subheading 3.2.2.1 ( see Note 5 ).
3.3. PIA Detection by Immunological Assays
3.3.1. Generation of PIA Antisera
1. Isolate PIA as described in Subheading 3.2.2 .
2. To produce PIA antiserum, immunize rabbits with 2 mg of PIA using a standard
protocol.
3. Dilute PIA antiserum in TBS buffer (1:100).
4. Absorb antiserum with the following extracts of a PIA-negative staphylococcal
mutant strain (e.g., S. epidermidis 1457 M10) for 16 h to reduce non-specific
binding. Prepare all extracts from cells of 50 mL of S. epidermidis 1457 M10
cultures or from 200 mL of bacterial supernatant, respectively:
a. Isolate extract by boiling cells with 1 mL 0.5 M EDTA for 5 min at 100 °C.
b. Isolate extract by boiling cells in 1 mL 1% sodium dodecyl sulfate (SDS) for
5 min at 100 °C.
c. Isolate extract isolated from cells treated with 1 mL lysostaphin.
d. Isolate 1 mL crude cell extract prepared by breaking cells with glass beads.
e. Isolate extract obtained from 200 mL culture medium precipitated by
trichloroacetic acid and resolve dry pellets in 1 mL PBS.
5. Sediment precipitated material by centrifugation (30 min, 28,000 × g , 4 °C) and
add1m M of sodium azide to the clear supernatant that will be used for further
investigation.