Technology of the month

Immunoprecipitation-based method for rapid isolation of intact lysosomes from mammalian cells with stable expression of a tagged lysosomal marker



Protocols / Fluorescence labeling & Protein Biochemistry

Cell lines / Stable

Main applications

Prepared lysosomes can be subjected to downstream biochemical analyses such as proteomics, metabolomics or simple Western blotting. This technology and downstream analyses could be used for basic lysosomal studies, e.g. to compare lysosomal composition and interactions after different treatments and under different physiological conditions, or to study the downregulation of different receptors, including adhesion GPCRs.

The technology

The first step is to generate the HEK293 cell line stably expressing a lysosomal protein TMEM192 tagged with HA or Flag. The authors generated these cell lines using a lentiviral vector. Stably transduced clones were selected using the antibiotic blasticidin. During the Lyso-IP procedure, such cells are mechanically lysed in the absence of detergents. The lysates are then incubated with commercially available magnetic beads coated with an anti-HA or anti-Flag antibody. The beads are washed in high salt buffer to remove unspecifically bound proteins and eluted with appropriate buffer to lyse the lysosomes. The quality of the preparation is assessed by Western blotting using antibodies against lysosomal (e.g. Lamp1, Cathepsin D) and other markers (e.g. citrate synthase for mitochondria, tubulin for cytosol, calnexin for endoplasmic reticulum, GM130 for Golgi, EEA1 for early endosome, etc.)..


For stable cell line generation*:

  • HEK293 cell line
  • Packaging lentiviral vectors
  • Transfer lentiviral vector with TMEM192-Flag or TMEM-HA inserts
  • Transfection reagent (Lipofectamine)
  • Polybrene
  • Facility for work with viruses
  • Blasticidin (selection antibiotic)
  • Cell culture room, reagents and buffers (DMEM and OptiMEM media, foetal bovine serum, trypsin, phosphate buffer saline, plastic consumables)

* The two stable lines generated by us can be disseminated upon request

For Lyso-IP:

  • Cell scraper
  • Bounce homogenizer
  • PBS, KPBS lysis buffer, protease and phosphatase inhibitors, NaCl, elution buffer (2% SDS, 50 mM Tris-HCl pH 7.6, 150 mM NaCl)

For testing the quality of the prep

  • Antibodies against lysosomal and other organellar markers
  • Western blotting tools and reagents

Background & Future perspectives

This technology was developed by Abu-Remaileh et al. for rapid isolation/enrichment of the intact lysosomes from the mammalian cells. The authors have set up this method in collaboration with Dr. Anita Krisko from the University Medical Center Goettingen in Germany. The authors are willing to share a detailed protocol upon request, or to collaborate with interested researchers.

Contact person

Katarina Trajkovic (Group leader)
Mediterranean Institute for Life Sciences (MedILS)
Split, Croatia

Links to webpages


Abu-Remaileh M, Wyant GA, Kim C, Laqtom NN, Abbasi M, Chan SH, Freinkman E, Sabatini DM. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes. Science. 2017 Nov 10;358(6364):807-813. doi: 10.1126/science.aan6298. Epub 2017 Oct 26. PMID: 29074583; PMCID: PMC5704967.



Do you want your technology to be the next highlighted here? Please contact and/or

Go back