Whole Cell Protein and LPS Gels
Categorized: Protein Purification and Gel Electrophoresis
Hitchcock and Brown. 1983. Journal of Bacteriology, 154:269-277
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.
- Lysing buffer:
- 2% SDS
- 4% 2-mercaptoethanol
- 10% glycerol
- 0.1 M Tris-HCl, pH 6.8
- Grow the bacteria overnight on appropriate agar plates.
- 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).
- Remove 1.5ml of the suspension and centrifuge in a microfuge for 1 1/2 minutes to pellet the cells.
- Solubilize the pellets in 100ul of lysing buffer.
- Heat the lysates to 100oC for 10 minutes.
- For protein digestion of the samples, add 25ug of proteinase K, solubilize in 10ul of the lysing buffer to each boiled lysate. Incubate at 60oC for 1 hour.
- Run the gel as usual and stain with silver (see the silver staining protocol).