Whole Cell Protein and LPS Gels

Reference:

Hitchcock and Brown. 1983. Journal of Bacteriology, 154:269-277

Objective:

This is a procedure analyzing LPS produced by bacteria, by using a protein-free LPS preparation and after gel electrophoresis, staining the gel using a LPS specific stain.

Materials:

• Lysing buffer:
• 2% SDS
• 4% 2-mercaptoethanol
• 10% glycerol
• 0.1 M Tris-HCl, pH 6.8

Method:

1. Grow the bacteria overnight on appropriate agar plates.
2. Harvest cells with A sterile loop and suspend in 0.9% saline to an OD₆₀₀ of 0.4 (equivalent to 200 Klett units using a blue filter).
3. Remove 1.5ml of the suspension and centrifuge in a microfuge for 1 1/2 minutes to pellet the cells.
4. Solubilize the pellets in 100ul of lysing buffer.
5. Heat the lysates to 100^oC for 10 minutes.
6. For protein digestion of the samples, add 25ug of proteinase K, solubilize in 10ul of the lysing buffer to each boiled lysate. Incubate at 60^oC for 1 hour.
7. Run the gel as usual and stain with silver (see the silver staining protocol).