Griffitts:LPS Analysis

Introduction

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

Procedure

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.

• 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

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.

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

Materials

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)

• 30.3 g Trizma base (Tris)
• 144.2 g glycine
• 10 g sodium dodecyl sulfate (SDS)

QS to volume with ddH₂0

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

• 20 mg/mL Proteinase K (in freezer)
• 20 mM Tris pH 8.0

Store at -20°C

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:

• 75 μL 100X SMAJ
• 75 μL 100X MCBT
• 75 μL 30% sucrose

Autoclave

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

References

^‡ Adapted from:

Campbell et al. (2003) "Striking complexity of lipopolysaccharide defects in a collection of Sinorhizobium meliloti mutants." J Bacteriol, 185:3853–62 PMID 12813079

Tsai, C.M., and Frasch, C.E. (1982) "A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels." Anal Biochem 119:115–119 PMID 6176137.