I am working on the Chaperone mediated autophagy and presently working over the isolation of lysosome from HEK cells. I am able to isolate the lysosomes but their inetegirty is very low when it is checked using beta-hexosaminidase assay. Kindly suggest how to increase the intergity of lysosomes.
Hello Pushpender Bhardwaj
You may use the Lyso-IP method. With the Lyso-IP technique, intact lysosomes are immunoprecipitated via epitopes that are added to the cytosolic portion of lysosomal transmembrane proteins. The fusion of triple human influenza virus hemagglutinin (3×HA) epitope tags to the lysosomal transmembrane protein 192 (TMEM192), TMEM192-3×HA, allow for the rapid isolation of lysosomes by using anti-HA magnetic beads. Remarkably, this takes only approximately ten minutes to accomplish. The isolated lysosomes can retain the lysosomal protease activity and also contain many lysosomal luminal proteins.
Additionally, the rapid timeframe of the TMEM192-tagged Lyso-IP method allows potentially labile molecules, such as the amino acids and other metabolites to remain stable for downstream analysis. Lysosomes isolated by the Lyso-IP method can be successfully used for a number of analysis such as proteomic, lipidomic, and metabolomic analysis.
Please refer to the references below.
1. https://www.science.org/doi/abs/10.1126/science.aan6298
2. https://pubs.acs.org/doi/abs/10.1021/acs.jproteome.9b00580
Best.
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