Quorum Sensing

5. Immunocomplexes are captured by adding 100μl 4 Fast Flow
   beads and incubating overnight at 4C with gentle rocking on
   a rocker/orbital shaker. 4 Fast Flow beads are collected by
   gentle pulse centrifugation (5 s at 14,000
   g). Discard the
   supernatant and wash the beads three times in ice-cold 1
   PBS
   pH 7.6 with centrifugation at 14,000
   gfor 20 s.


6. Following the final wash, 4 Fast Flow beads are resuspended in
   Laemmli sample buffer, boiled for 5 min at 95C, and then
   collected by centrifugation at 14,000
   gfor 20 s. The result-
   ing supernatant is then loaded on polyacrylamide (e.g., 4–12%
   or 8–16%) for SDS-PAGE. Samples can be stored frozen in
   fresh centrifuge tubes at 20 C but must be re-boiled for
   5 min immediately prior to loading onto a gel.

3.4 SDS-PAGE
and Immunoblotting

1. Protein concentration of whole cell lysates should be predeter-
   mined as stated in Preparation of Cell Lysates methods.


2. Samples are diluted in Laemmli’s sample buffer at equal protein
   concentrations, heated at 95C for 5 min, and then loaded on
   4–12% or 8–16% SDS-polyacrylamide gels and run at standard
   voltage. Following electrophoresis, gels can proceed to immu-
   noblotting or staining with Coomassie Blue for further in-gel
   digestion and mass spectrometric characterization of proteins/
   proteomes [17].


3. For immunoblotting, separated proteins are electrophoretically
   transferred to a PVDF membrane and quality of the transfer is
   monitored by Ponceau S staining.


4. PVDF membrane is washed twice with distilled water (or 1
   
   PBS) and then incubated for 1 h at room temperature in
   blocking buffer (5% nonfat milk in 1
   PBS pH 7.6, containing
   0.18% Tween 20) to prevent nonspecific binding.


5. Desired primary Abs (seeSubheading2) are diluted in blocking
   buffer, added to the membrane, and incubated overnight at
   4 C with gentle rocking on a rocker/orbital shaker. As an
   example, we have used anti-ZO-3 and anti-JAM-A antibodies
   diluted 1:1000 in blocking buffer (Fig.2a). Appropriate dilu-
   tions for primary antibodies should be empirically determined.
   Routinely, anti-GAPDH Abs should be included as a loading
   control.


6. Following overnight incubation, wash the PVDF membrane in
   1
   PBS pH 7.6 with 0.18% Tween 20 and then incubate with
   IRDye 800CW or IRDye 680CW secondary Abs diluted
   1:10,000 for 1 h at room temperature. If using other detection
   Abs, the appropriate dilutions should be empirically
   determined.

Autoinducer Effects on Mammalian Cells 221