Quorum Sensing

6. Resolve the pellet in 50 mM Tris–SDS buffer. Measure the
   protein concentrations using the Bradford assay according to
   the manufacturer’s protocol.

3.2.2 Intracellular Protein
Preparation

1. Centrifuge the individual 500 ml culture at 7000g for
   30 min at 4C(seeNote 12).


2. Remove the supernatant. Resuspend the cell pellet in ice-cold
   10 ml Tris–PI buffer.


3. Lyse the cells by sonication first and then pass them through
   French Press Cell twice (seeNote 13). Check the lysate under
   the microscope to ensure the complete lysis.


4. Remove the cell debris by centrifugation at 4000g for
   15 min at 4C.


5. Separate the cell membrane proteins from soluble intracellular
   proteins by ultracentrifugation at 80,000gfor 1 h at 4C.
   SeeSubheading3.2.3to continue with the membrane protein
   (the pellet after ultracentrifugation) extraction.


6. Carefully take out the supernatant and add 6 volumes of pre-
   chilled acetone to precipitate the intracellular proteins. Mix and
   incubate the solution at 20 C overnight.


7. Centrifuge (20,000g, 30 min, 4C) to collect precipitated
   proteins.


8. Discard the acetone supernatant. Air-dry the pellet. Dissolve
   the pellet in Tris–SDS buffer and determine the protein con-
   centration by Bradford assay.

3.2.3 Cell Membrane
Protein Preparation

1. After ultracentrifugation (seeSubheading3.2.2,step 5), gently
   wash the pellet with Tris–PI buffer to remove the remaining
   intracellular proteins.


2. Pellet the protein again with ultracentrifugation (the same
   setting as Subheading3.2.2,step 5).


3. Carefully remove the supernatant. Dissolve the proteins in
   100 mM Tris–HCl, pH 7.5 with 2% (wt/vol) SDS. Determine
   the protein concentration.

3.3 SDS-PAGE and
Gel Staining

1. Load 2μg of protein of each sample on 15% SDS-PAGE gel (see
   Note 14). Run the electrophoresis according to the standard
   protocol [25] until the front dye just migrates off the gel.


2. After electrophoresis, carefully open the gel cassette and take
   out the running/resolving gel. Gently rinse the gel with deio-
   nized water. Stain the gel with colloidal blue staining buffer
   with very gentle horizontal agitation for 2–12 h [26].


3. To remove the excessive stain, soak the gel in deionized water
   with gentle shaking. Change water every 30 min for a few times
   until distinct protein bands appear [26].

GeLC-MS/MS Proteomics to Identify Quorum Sensing Targets 197