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 100 µl of lysing buffer.
5. Heat the lysates to 100ºC for 10 minutes.
6. For protein digestion of the samples, add 25 µg of proteinase K, solubilize in 10 µl of the lysing buffer to each boiled lysate. Incubate at 60ºC for 1 hour.
7. Run the gel as usual and stain with silver (see the silver staining protocol).