LYSOSOMAL ISOLATION PROTOCOL
Scott Vermilyea
Published: 2024-06-26 DOI: 10.17504/protocols.io.kqdg32kdqv25/v1
Abstract
This protocol details the isolation of lysosomes.
Steps
Subcellular Lysosome Isolation Protocol
1.
In Vitro Harvest
1.1.
Following designated treatment, wash cells three times with DMEM followed by the application of 0.5mL of homogenization buffer supplemented with proteinase and phosphatase buffer.
Homogenisation Buffer:
| A | B |
|---|---|
| Sucrose | 250 mM |
| EDTA | 2 mM |
| Magnesium chloride | 1.5 mM |
| Potassium Chloride | 10 mM |
| HEPES | 20 mM |
1.2.
Gently detach cells with cell scraper.
1.3.
Homogenize using Teflon homogenizer (12 strokes).
1.4.
Separate 50µL of total homogenate (TH) and lyse with *TNE lysis buffer.
TNE lysis buffer:
| A | B |
|---|---|
| Tris | 50 mM |
| NaCl | 150 mM |
| EDTA, pH 7.4 | 5 mM |
| SDS | 1% |
| NP-40 | 0.5% |
| DOC | 0.5% |
| protease/phosphatase inhibitors |
1.5.
Centrifuge remaining volume of TH at 1000x g,4°C, collect the supernatant.
1.6.
Further centrifuge the supernatant for 20000x g,4°C to collect the precipitate as crude lysosomal fraction (CLF). Lyse the CLF with *TNE lysis buffer, and prepare lysates for western blot.
TNE lysis buffer:
| A | B |
|---|---|
| Tris | 50 mM |
| NaCl | 150 mM |
| EDTA, pH 7.4 | 5 mM |
| SDS | 1% |
| NP-40 | 0.5% |
| DOC | 0.5% |
| protease/phosphatase inhibitors |
