Quorum Sensing

3.4 In-Gel Digestion 1. Excise individual sample lanes; cut the entire lane into ten gel
bands, namely ten fractions (Fig.1). Transfer each gel fraction
into a Protein LoBind tube (seeNote 15).

2. Destain each gel fraction in destaining solution at 37C for
   30 min (seeNote 16). Change the destaining solution and
   incubate another 15 min. Repeat this step a couple of times
   until the gel slices lose the blue color.


3. Remove the destaining solution. Shortly wash each gel fraction
   once with acetonitrile and discard the solvent.


4. Incubate each gel fraction in acetonitrile for 5 min. Then
   remove and discard the solvent.


5. Add DTT solution to soak the gel slices and incubate the tubes
   at 60C for 1 h. Remove DTT solution.


6. Wash the gel fractions again with acetonitrile.


7. Add IAA solution and incubate the tubes for 30 min in the dark
   (seeNote 17). Then remove IAA solution.


8. Wash the gel fractions twice with 50 mM (NH 4 )HCO 3.


9. Add 80% acetonitrile and incubate the tubes at 37C for 1 h
   (see Note 18). Discard the acetonitrile solution after
   incubation.


10. Dry the gel slices completely by incubating at 50C or using
    SpeedVac Concentrator.


11. Add 100μl trypsin solution (1μg) per gel fraction/per tube
    and incubate at 37C for 20 min (seeNote 19). Then top up
    each gel fraction with 200μl trypsin buffer and incubate at
    37 C at least 4 h or overnight.


12. Add 300μlH 2 O and 65μl 10% formic acid to each digest (see
    Note 20).

3.5 Peptide
Extraction Using
ZipTip®C18 Resin
Pipette Tips

Treat each sample fraction/each tube with the following

procedure.

1. Use one 10 μl pipette to take one ZipTip. Aspirate 10μl
   wetting solution and dispense to the waste. Repeat once.


2. Wash the tip with 10μl washing solution twice. Aspirate 10μl
   washing solution and dispense to the waste.


3. Load the resin with peptides by pipetting up and down many
   times in the sample fraction digest.


4. Wash the loaded resin three times with the washing solution.


5. Take a new Protein LoBind tube and add 50 μl elution
   solution.


6. Elute the peptides in the 50μl elution solution by pipetting up
   and down at least ten times.

198 Yilei Liu et al.