Jun 25, 2024
LYSOSOMAL ISOLATION PROTOCOL
- Scott Vermilyea1
- 1Univ. of Minnesota, Team Lee
Protocol Citation: Scott Vermilyea 2024. LYSOSOMAL ISOLATION PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32kdqv25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 07, 2024
Last Modified: June 25, 2024
Protocol Integer ID: 102392
Keywords: Crude lysosomal fraction, Homogenisation , Precipitation, Lysis buffer
Funders Acknowledgement:
ASAP
Grant ID: 000592
Abstract
This protocol details the isolation of lysosomes.
Materials
Homogenisation Buffer:
| A | B | |
| Sucrose | 250 mM | |
| EDTA | 2 mM | |
| Magnesium chloride | 1.5 mM | |
| Potassium Chloride | 10 mM | |
| HEPES | 20 mM |
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 | ||
Subcellular Lysosome Isolation Protocol
Subcellular Lysosome Isolation Protocol
30m
In Vitro Harvest
Following designated treatment, wash cells three times with DMEM followed by the application of 0.5 mL 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 |
Gently detach cells with cell scraper.
Homogenize using Teflon homogenizer (12 strokes).
Separate 50 µL 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 | ||
Centrifuge remaining volume of TH at 1000 x g, 4°C, 00:10:00 , collect the supernatant.
10m
Further centrifuge the supernatant for 20000 x g, 4°C, 00:20:00 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 | ||
20m