This seems to be the only kit (made by Invent Biotechnologies) that's based on a non-density gradient and ultracentrifugation method. Has anyone used it successfully to isolate lysomes from cultured cells (eg skin fibroblasts)? Or can you recommend something else to purify lysosomes?
Hi, may be you can use following steps.
1. breakdown the cell membrane
2. low speed centrifuge to get supernatant.
3. high speed centrifuge to get pellet
4. do Percoll gradient
5. check lysosome fraction in which gradient
Lysosomes are spherical vesicles in eukaryotic cells that are responsible for waste removal. The digestive enzymes contained in lysosomes play a vital role in digesting excess or worn-out organelles, food particles and engulfed viruses or bacteria. Lysosomes are relatively large organelles ranging in size from 0.1 to 1.2 um. The ability to isolate lysosomes is an important first step in studies of autophagy, protein degradation and protein recycling in a cell. Traditional methods for isolating lysosomes are based on density-gradient ultracentrifugation. A large amount of starting material is required and the methods are tedious and time-consuming and contain significant cross-contamination. Currently, all commercial kits for lysosome isolation are based on methods developed in the 1970s. Unlike any other lysosome isolation kit on the market, our kit employs a patented spin-column-based technology that is simple, rapid and efficient. The amount of starting cells/tissues required is much smaller than that of traditional methods. This kit can significantly enrich lysosomes from cultured cells or tissues without using a Dounce homogenizer and ultracentrifugation. The whole protocol can be done in less than 1.5 hours.
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