Protocol name: LPS detection
Prepared by: Monica Ponder
Date Created/Revised: 5/27/03
Purpose: convert polysaccharides to
alditol acetates detected by GC/MS
Reference(s): Mindock et. Al
Comments: *Only glass pipettes and containers may be used.*
*All caps on scintillation vials must have a Teflon liner or organic compounds
will extract the lipids from the plastic and contaminate your samples!!!
1. 150 ml cultures OD 600= 0.4 Centrifuge cells at 10, 000 x g and resuspend in
60 mls MilliQ. Transfer cells: MilliQ mix to 500 ml baked Erlenmeyer flask.
2. Add 130 ml chloroform and 15 ml methanol ( both HPLC grade)
3. Placed in shaker at 30°C overnight.
4. Using separatory funnel (glass) remove the bottom organic layer containing the
lipids into new naked Erlenmeyer flask. The water layer should be placed in
original flask and the extraction process repeated to remove the majority of the
lipids.
5. Dry water layers down by roto- vap to volume of 4 mls
6. added 10 units of DNase ( 1U/ ul), 10 units of RNAase ( 1U/ ul), 100 ul of 100
mg/ml MgCl2
7. Incubate 4 hours at room temperature
8. Add 2 volumes of ETOH (99%, -20°C) and centrifuge at 10, 000 x g for one hour.
Must use Teflon bottles!!!!!
9. Decanted ETOH and allowed pellet to dry thoroughly before using 5 mls MgCl2
(1mg/ml): MilliQ to resuspend pellet
10. Using Spectrapor tubing 32 mm width, 20.4 mm diameter MW 12-14,000
dialyzed for 3 days at 24C changing water daily
11. Next must lyophilize contents of the dialysis bag for 36 hours ( place in 2 dram
glass scint vials)
12. 2ml of 2M TriFluoroacetic acid (Spectrum, TR119) added and sonicated 5
minutes in sonicating bath
13. Placed at 120 C for 2 hours sonicating every 30 minutes with 10 minutes to cool
before sonicating so don’t crack vial
14. Dried down with N evaporator
15. Extract with 1:1 Chloroform and hexane (95% pure), shake vigorously, add 1 ml
milliQ. Shake vigorously 5 minutes
16. Using a Pasteur pipette that has been pulled so it has a thin tip remove the bottom
organic layer to a new 2 dram scint vial with Teflon lined cap
17. Second chloroform hexane extraction performed on water layer and decanted
organic layer to scint vial containing previous organic layer
18. dry down both organic and water layers on N2 evaporater
19. Add 0.5 ml MilliQ to the water layer ( top layers) to remove last traces of TFA
20. Blow to dryness
21. Made NaBorohydride solution: 0.2mg to 100 ul MilliQ. Add 2 drops from
Pasteur pipette into water layers. Should see a visible fizzing. If no fizz add more
drops. Next add 300 ul to remaining NaBorohydride solution in test tube mix and
add 6 drops of this to water layers. Incubate overnight at room temperature.
22. Add 6 drops HCl to water layer and incubate 2 hours room temp. dry down by
evaporation overnight or use N evaporator
23. Resuspend in 0.3 ml milliQ making sure all carbohydrates are resuspended by
flicking sides gently. Evaporate again
24. Resuspend in 4 drops glacial acetic acid followed by 4 drops of methanol mixing
thoroughly by flicking. Dried down.
25. Resuspend in 1 ml methanol, dry down, repeat 3 times to completely remove
proteins and contaminants of carbohydrates.
26. Sugars are now cleaved and washed so must convert to alditiol acetates. Also
create a control using 3 drops of pure glycerol that is treated same as all samples
27. Add 0.3 mls of pyridine, sonicate 10 minutes in sonicating bath
28. Add 0.1 ml of acetic anhydride, sonicated 5 minutes in bath
29. Reaction to go 24 hours at room temperature then dried down
30. Chloform: water extraction (3:1). Added 1.5 mls CHCl3 and 0.5 mls MilliQ to
glass scint vials then shaken vigorously for 5 minutes.
31. Decanted bottom organic layer to new 2 dram glass scint vial with Teflon cap
using a long thin Pasteur pipette. These contain the alditol acetates that are
analyzed by GC:MS