ReadiPrep™ Lipopolysaccharide (LPS) Isolation Kit

1. Grow bacteria on the LB agar plate.

2. Add LPS isolation buffer to the bacterial pellet. Sonicate the mixture, then incubate it on ice for 10 minutes.

3. Add proteinase K to the bacterial lysate and incubate the mixture at 60°C for one hour.

Thaw all the kit components at room temperature before starting the experiment. 

1. Grow the bacteria on an LB agar plate overnight at 37°C.

2. The next day, collect bacterial colonies in ice-cold PBS. Measure the optical density (OD) at 600 nm using a spectrophotometer. Ensure the OD at 600 nm is greater than 0.6.

3. Centrifuge the tube at 2500g for 10 minutes to collect the pellet. After removing the supernatant, centrifuge again at 2500g for 10 minutes to ensure all the supernatant is removed.

4. Measured the weight of the pellets.

   Note: For an E. coli suspension with OD > 0.6, the pellet weight should be greater than 10 mg.

5. Add 200 µL of isolation buffer to the LPS pellet. Use 200 µL for a 20 mg pellet.

6. Sonicate the pellet three times for 30 seconds each at 10 watts. Then, incubate the tube on ice for 10 minutes.

7. Centrifuge the tube at 2500g for 10 minutes at 4°C.

8. Transfer the lysate into a new, clean tube.

9. Add Proteinase K to the lysate at a concentration of 0.1 mg/mL.

   Note: For example, if you have 200 µL of lysate, add 1 µL of 20 mg/mL Proteinase K solution.

10. Incubate the tube at 60 °C for one hour.

11. Centrifuge the tube at 2500g for 10 minutes at 4°C.

12. Collect the supernatant.

13. LPS can be measured using carbohydrate detection methods. Alternatively, its purity can be assessed by running it on an SDS-PAGE gel and then staining it with Coomassie blue.