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Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit
Published on: April 19, 2019
Rapid isolation of lysosomal membranes from cultured cells
D Mušálková1, J Lukáš, F Majer
1Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic.
Abstract:
We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.
Insights
Researchers developed a simple method to isolate lysosomal membranes using methionine methyl ester, achieving a 30-fold enrichment. This robust technique minimizes contamination and preserves lysosomal vacuolar ATPase activity.
Area of Science:
- Cell Biology
- Biochemistry
- Membrane Biology
Background:
- Lysosomes are crucial organelles involved in cellular degradation.
- Efficient isolation of pure lysosomal membranes is essential for studying their function.
- Existing methods may involve complex procedures or non-physiological cell treatments.
Purpose of the Study:
- To develop a simple, efficient, and robust method for enriching lysosomal membranes.
- To characterize the purity and integrity of the isolated lysosomal membranes.
- To provide an alternative to existing lysosomal membrane isolation techniques.
Main Methods:
- Selective lysosomal disruption using methionine methyl ester in HEK293 and HeLa cells.
- Isolation of organelle concentrate from postnuclear supernatant.
- Separation of lysosomal membranes on a step sucrose gradient.
- Analysis of purity and functional integrity (vacuolar ATPase activity).
Main Results:
- A 30-fold enrichment of lysosomal membranes was achieved with over 8% yield.
- The method effectively eliminated mitochondrial and other cellular contaminants.
- Isolated lysosomal membranes retained functional vacuolar ATPase activity.
- The procedure is robust across tested cell lines and avoids superparamagnetic particle overloading.
Conclusions:
- Methionine methyl ester-based lysosomal disruption provides a simple and effective method for lysosomal membrane enrichment.
- The isolated membranes are pure, functional, and suitable for further biochemical studies.
- This method offers a valuable alternative for lysosomal membrane isolation, adaptable to various cell types.
