Griffitts:LPS Analysis

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Contents

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

Store at -20°C

Sample Buffer (5 mL)

SMM Sucrose (75 mL)

To 75 mL sterile water agar add:

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.
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