Quorum Sensing

then immediately lysed with ice-cold cell lysis buffer (seeSub-

heading2). 1 ml cell lysis buffer per 10^7 cells in a 150 cm^2 flask,

or 200–300μl cell lysis buffer per well in a 6-well plate is

acceptable.

3. While still on ice, scrape cells in lysis buffer off the growing
   surface using a rubber scraper and transfer to a centrifuge tube.
   The cell suspensions are homogenized through a 21-gauge
   needle and then centrifuged at 14,000
   gfor 30 min at
   4 C. Resulting supernatant is collected, aliquoted into fresh
   centrifuge tubes, and stored at  70 C until ready to be
   assessed.


4. Protein concentrations of the cell lysates is determined using a
   detergent compatible colorimetric assay (seeNote 4); average
   protein concentration has previously been determined to be
   around 5000–7000μg/ml.


5. Whole cell lysates are diluted in Laemmli’s sample buffer at
   equal protein concentrations, heated at 95C for 5 min, and
   then subjected to SDS-PAGE.


6. Alternative: whole cell lysates are immunoprecipitated prior to
   loading on a gel.

3.3 Immuno-
precipitation

1. Prepare Protein G Sepharose 4 Fast Flow beads (GE Health-
   care, Uppsala, Sweden) by washing the beads twice in 1
   PBS
   and then restoring to a 50% slurry in 1
   PBS. Take care to
   avoid disruption of the beads.


2. Samples are pre-cleared by combining 100μl of 4 Fast Flow
   beads (50% bead slurry) with 1 ml of whole cell lysates and
   incubating for 30–60 min at 4C on a rocker/orbital shaker.
   Pre-clearing samples will aid in reducing nonspecific binding of
   proteins to the beads in succeeding steps. Remove 4 Fast Flow
   beads by centrifugation at 14,000
   gfor 10 min as per the
   manufacturer’s instructions. Transfer the resulting supernatant
   to a new centrifuge tube.


3. The protein concentration of the cell lysates should be prede-
   termined as described in Preparation of Cell Lysates methods.
   Dilute the whole cell lysates to approximately 1μg/μl total
   protein in 1
   PBS. If necessary, a more concentrated cell
   concentrate (e.g., 10μg/μl) may be required to immunopre-
   cipitate found at low concentrations.


4. Samples with equivalent protein concentrations are immuno-
   precipitated by combining 500μl of cell lysate with primary
   Abs and incubating overnight at 4C with gentle rocking on a
   rocker/orbital shaker. As an example, we have used anti-ZO-3
   and anti-JAM-A Abs (Fig.2a). Appropriate dilutions for pri-
   mary antibodies should be empirically determined.

Autoinducer Effects on Mammalian Cells 219