Griffitts:LPS Analysis

Introduction
This method‡ allows resolution of different species of both smooth and rough LPS.

Purification

 * Grow cells to late log phase in SMM-sucrose
 * Centrifuge 1 mL for 1 minute at 13,200 rpm
 * Resuspend in 100 μL of lysis buffer
 * Incubate at 100°C for 10 min
 * Add 60 μg of proteinase K
 * Incubate at 60°C for 1 h
 * Add 20 μL of sample buffer
 * Boil for another 5 min

Electrophoresis
NOTE: To avoid staining artifacts, the gel casting tray and staining dishes should be thoroughly washed with detergent, rinsed in dH2O, and wiped with ethanol-soaked Kimwipes. NOTE: Tsai and Frasch recommend incorporating 4 M urea into the gel; Campbell et al. recommend replacing 0.2 M glycine with 0.1 M tricine-sodium in the gel and running buffer.
 * Load 2.5 μL of the LPS preparation onto a standard SDS-acrylamide minigel containing 15% acrylamide
 * Electrophorese in 1X Laemmli running buffer at 20 mA until the bromophenol blue has migrated 10 cm

Staining

 * Place the gel in 200 mL fixation buffer overnight
 * Replace the fixation buffer with 200 mL oxidation buffer
 * Shake at 40 rpm for 5 minutes
 * Wash three times in 500–1000 mL ddH2O on a shaker at 40 rpm for 15 minutes (use a new dish)
 * Carefully discard the water
 * Add 150 mL staining solution
 * Shake at 70 rpm for 10 minutes
 * Wash three times in 500–1000 mL ddH2O on a shaker at 40 rpm for 15 minutes (use a new dish)
 * Carefully discard the water
 * Add 200 mL developer solution
 * Shake at 45 rpm for 2–5 minutes
 * NOTE: Stop when the stain reaches the desired appearance or when discoloration begins to affect the gel background
 * Wash three times in 500–1000 mL ddH2O on a shaker at 40 rpm for 15 minutes (use a new dish)
 * Store in ddH2O

Developer Solution (1 L)

 * 995 mL ddH2O
 * 50 mg citric acid
 * 500 μL 37% formaldehyde

Fixation Buffer (200 mL)

 * 110 mL ddH2O
 * 80 mL 100% ethanol
 * 10 mL glacial acetic acid

10X Laemmli running buffer (1 L)
QS to volume with ddH20
 * 30.3 g Trizma base (Tris)
 * 144.2 g glycine
 * 10 g sodium dodecyl sulfate (SDS)

Lysis Buffer (10 mL)

 * 8.85 mL ddH2O
 * 1 mL 1 M Tris-HCl (pH 6.8)
 * 150 mg sodium dodecyl sulfate (SDS)
 * 150 μL β-mercaptoethanol

Oxidation Buffer (200 mL)

 * 110 mL ddH2O
 * 80 mL 100% ethanol
 * 10 mL glacial acetic acid
 * 1.4 g periodic acid

Proteinase K
Store at -20°C
 * 20 mg/mL Proteinase K (in freezer)
 * 20 mM Tris pH 8.0

Sample Buffer (5 mL)

 * 575 μL ddH2O
 * 3.125 mL 80% glycerol
 * 1 mL 1 M Tris-HCl (pH 6.8)
 * 50 mg sodium dodecyl sulfate (SDS)
 * 250 μL β-mercptoethanol
 * 50 μL 2% bromophenol blue

SMM Sucrose (75 mL)
To 75 mL sterile water agar add: Autoclave
 * 75 μL 100X SMAJ
 * 75 μL 100X MCBT
 * 75 μL 30% sucrose

Staining Solution (150 mL)
NOTE: This should be made fresh for each use and then discarded (it may become explosive if stored) NOTE: Use a stir bar
 * Add 2 mL of concentrated ammonium hydroxide to 28 mL of 100 mM sodium hydroxide
 * Add 5 mL 20% silver nitrate
 * NOTE: A brown precipitate will briefly form and then disappear
 * Add 115 mL ddH2O