Detection of glycolipids in plasma membrane fractions

Detection of glycolipids in plasma membrane fractions

This protocol assumes that bacterial lysates were separated in a density gradient, and that membrane fractions were already collected. See references Morita et al. (2005) and Hayashi et al. (2016) for more details.

Materials:

1. Membrane fractions
2. Solvents        
   • chloroform
   • methanol 
   • 1-butanol 
   • water-saturated butanol 
   • chloroform/methanol/13 M NH₃/1 M NH₄Ac/water (180:140:9:9:23)
3. Vacuum Concentrator Centrifuge
4. Reacti-Vap™ Evaporator System (Thermo Scientific)
5. Water bath sonication system
6. High-performance thin layer chromatography (HPTLC) plates (Merck 5547)
7. TLC tank
8. TLC sprayer

Protocol

Extraction of glycolipids from plasma membrane fractions

1. Have the membrane fractions ready. If frozen, thaw them completely on ice.
2. Vortex your tube that contains membrane fractions until the sample is homogeneous, then: 
3. In duplicate and using 2-ml microtubes, add 1.4 ml of chloroform/methanol (1:1) to 210 µl of sucrose gradient fractions to make a final ratio of chloroform/methanol/water (10:10:3).
4. Let it sit at room temperature for 2 hours.

5. Spin for 2 min at 14,000 rpm on microfuge, and transfer and combine the supernatant of both, duplicate tubes into to a fresh tube.

6. Dry under nitrogen stream using Reacti-Vap evaporator. Set temperature at 60-70°C. Because of high concentrations of sucrose, solvent may not dry completely. 
7. Resuspend in 600 ml of water.  Add 1200 µl of water-saturated butanol to all tubes. 
8. Vortex, microfuge for 30 sec, and transfer the upper phase to a new tube.     
9. Add another 600 µl of water-saturated butanol and repeat the extraction, combining the upper phase to the previous existing one.
10. Add 600 µl of water to the combined butanol phase and repeat the extraction again. 
11. Transfer the upper phase, and dry the combined extractions on a vacuum concentrator centrifuge. 
12. Add 300 ml of water and 600 µl of water-saturated butanol to all tubes.     
13. Vortex, microfuge for 30 sec, and transfer the upper phase to a new tube.     
14. Add another 300 µl of water-saturated butanol and repeat the extraction. 
15. Dry the final combined butanol phase on a vacuum concentrator centrifuge.

16. Resuspend the dried sample in 25 µl of water-saturated butanol. Vortex well and sonicate briefly in a water bath sonicator to resuspend the pellet.     

Thin layer chromatography

17. Equilibrate TLC tank with ~50 ml of chloroform/methanol/13 M NH₃/1 M NH₄Ac/water (180:140:9:9:23). Spot 6 - 9 ml of samples on an HPTLC plate and develop for ~45 min. Spot 1.5 µl at a time, dry, and apply again until applying the desired sample volume. Adjust the loading volume according to the lipids you want to detect. 

18. Dry the plate at room temperature (RT), and spray using a TLC sprayer:

A). Orcinol reagent (stains PIMs and other glycolipids), and develop at 100°C for 5 min or longer. 

B). Cupric acetate (general charring of all organic carbons), and develop at 150°C for 10-20 min.

C). Iodine vapor (reversible staining of organic carbon double bonds). Stain at RT for 10-20 min. 

D). Molybdenum Blue reagent (stains phospholipids) and develop at RT for 10 min or longer. 

References

García-Heredia, A., Kado, T., Sein, C. E., Puffal, J., Osman, S. H., Judd, J., . . . Siegrist, M. S. (2021). Membrane-partitioned cell wall synthesis in mycobacteria. Elife, 10. doi:10.7554/eLife.60263

Morita, Y. S., Patterson, J. H., Billman-Jacobe, H., & McConville, M. J. (2004). Biosynthesis of mycobacterial phosphatidylinositol mannosides. Biochemical Journal, 378(2), 589-597.

Morita, Y. S., Velasquez, R., Taig, E., Waller, R. F., Patterson, J. H., Tull, D., . . . McConville, M. J. (2005). Compartmentalization of lipid biosynthesis in mycobacteria. J Biol Chem, 280(22), 21645-21652. doi:10.1074/jbc.M414181200

Hayashi, J. M., Luo, C. Y., Mayfield, J. A., Hsu, T., Fukuda, T., Walfield, A. L., . . . Morita, Y. S. (2016). Spatially distinct and metabolically active membrane domain in mycobacteria. Proc Natl Acad Sci U S A, 113(19), 5400-5405. doi:10.1073/pnas.1525165113

Recommended reading:

Deranieh, R. M., Joshi, A. S., & Greenberg, M. L. (2013). Thin-layer chromatography of phospholipids. Membrane Biogenesis: Methods and Protocols, 21-27.