Jan 11, 2026

Public workspaceRapid Isolation of Lysosomes (LysoIP) from mouse brain tissue

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzj414lx1/v1
  • Ali Ghoochani1,2,3,4,
  • Monther Abu-Remaileh1,2,3,4
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzj414lx1/v1
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Rapid Isolation of Lysosomes (LysoIP) from mouse brain tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj414lx1/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: October 14, 2024
Last Modified: January 13, 2026
Protocol Integer ID: 109774
Keywords: LysoIP, lysosomes isolation, mouse brain lysosomes, lysosomes from fresh mouse brain tissue, lysoip from mouse brain tissue, rapid isolation of lysosome, lysosome, assessing lysosomal protein profile, lysosomal protein profile, preparing lysoip, mouse brain tissue this protocol, proteomic analysis, fresh mouse brain tissue, lysoip, purification method, mouse brain tissue, using immunoprecipitation, purification, recycling of biomolecule
Abstract
This protocol outlines a method for the rapid isolation of lysosomes from fresh mouse brain tissue. Lysosomes serve as metabolic hubs for the degradation and recycling of biomolecules. Despite their critical cellular functions, quantitatively assessing lysosomal protein profiles has been challenging. To address this, we developed a rapid harvesting and purification method using immunoprecipitation (LysoIP). This protocol provides detailed instructions for preparing LysoIP from mouse brain tissue for proteomic analysis.
Materials
1x extraction buffer :
  • 50 mM HEPES, pH 7.4
  • 40 mM NaCl
  • 2 mM EDTA
  • 1% Triton X-100 (Sigma)
  • 1.5 mM sodium orthovanadate (NaVO4)
  • 30 mM sodium fluoride (NaF)
  • 10 mM sodium pyrophosphate (Na4P2O7)
  • 10 mM sodium β-glycerophosphate
  • Complete EDTA-free Protease Inhibitor Cocktail

Troubleshooting
Materials
Materials and Reagents

  1. Anti-HA magnetic beads: Thermo Fisher Scientific
  2. Optima LC/MS water
  3. Phosphate-buffered saline (PBS): pH ~7.4
  4. Complete EDTA-free Protease Inhibitor Cocktail: Roche
  5. Magnetic separation equipment: DynaMag Spin Magnet
  6. 1x extraction buffer (Material section)
  7. Centrifuge
  8. Homogenizer: 2 ml homogenizer 
Equipment Preparation
Wash the glass vessel homogenizer with MilliQ Water, 10 times each. wash the tissue grinder
homogenizer thoroughly with DI Water and MilliQ Water, especially the gap between the white
parts, don’t touch the part that goes into the glass vessel. Then dry upside-down using paper
towels. Carefully place the glass vessels against something to prevent falling down. Minimize
any contact between the grinder and anything else.

  • Pre-chill all equipment and buffers used for homogenization and immunoprecipitation to 4°C.
  • Set centrifuge temperature to 4°C for all centrifugation steps.
Prepare microcentrifuge tubes as follows on a metal rack on ice (for each sample, from left to
right):
➀ 2 mL tube for posthomogenization
➁ 1.5 mL tube for whole brain fraction
➂ 1.5 mL tube for beads
➃ 1.5 ml tube for 3rd wash
➄ 1.5 mL tube for final lysoIP samples

Preparation of Anti-HA beads
Pool all required beads volumes together (Amount100 µL / sample, e.g.Amount800 µL total for 8 samples, extra is not needed).
Wash 3 times with the same volume (Amount100 µL / sample, e.g.Amount800 µL total for 8 samples) of ice-cold PBS containing Complete EDTA-free Protease Inhibitor Cocktail. After each wash, place the tube on the magnet, aspirate the supernatant, and add the same volume of ice-cold PBS containing Complete EDTA-free Protease Inhibitor Cocktail.
After thirs wash, Resuspend beads with the same volume (Amount100 µL / sample, e.g.Amount800 µL total for 8 samples) of ice-cold PBS containing Complete EDTA-free Protease Inhibitor Cocktail.
Aliquot 100 μL into each ➂ 1.5 mL tube for beads.
Isolation of Lysosomes from Mouse Brain
Tissue Collection and Preparation:
Process each mouse brain separately to ensure rapid isolation of lysosomes.
Euthanize the mouse and transfer the whole brain into 2 ml homogenizer (on ice)
Homogenization
Critical: All steps of the procedure must be performed on ice 

Critical
Immediately add Amount950 µL ice-cold PBS containing Complete EDTA-free Protease Inhibitor Cocktail into 2 ml homogenizer.
Homogenize with 25 gentle strokes (on ice).
Transfer the homogenized tissue to a 2 ml tube.
Reserve Amount25 µL of the homogenate ( 2.5% of the total tissue) for processing as the whole brain fraction.

Recommended: Add 1 ml of ice-cold PBS with Protease Inhibitor Cocktail to the 2ml tube (Step 6.3) to minimize pellet contamination after centrifuge (next step).

Centrifuge atCentrifigation1000 x g, 00:02:00 at 4°C.




2m
Immunoprecipitation of Lysosomes
15m 25s
Transfer the supernatant containing cellular organelles to a 1.5 ml tube containing Amount100 µL of anti-HA magnetic beads

Incubate on a gentle rotator shaker for Duration00:15:00 at Temperature4 °C

15m
Place the tube on magnet. Count at least Duration00:00:25 to allow for beads to be pulled by
magnets.

Note: it is important to keep this count the same between each wash and each sample
for consistency I.e. 25 seconds each time .
25s
Wash the beads fraction three times with Amount1 mL PBS containing Protease Inhibitor Cocktail using a DynaMag Spin Magnet.

Note:
  • During the first wash, make sure to aspirate any liquid trapped on the inner side of the cap.
  • Pipet up and down 3 times and keep consistent each wash, each sample.


After the second wash, resuspend beads with PBS containing Protease Inhibitor Cocktail and then transfer it to the clean ➃ 1.5 ml tube for third wash (this step helps give cleaner results).
Place the tube on magnet wait Duration00:00:25 to allow beads to be pulled by magnets. Then, aspirate the supernatant.

25s
Lysosomal Protein Extraction
30m
Resuspend the beads with Amount80 µL of ice-cold 1x extraction buffer.
Incubate at least for Duration00:15:00 at Temperature4 °C
15m
After Duration00:15:00 finishing the last IP, place the IP samples on the magnet to separate the beads. Collect the supernatant and transfer it to a new 1.5 ml tube. Store the IP samples at Temperature-80 °C until further processing.

15m
For whole brain fraction add Amount160 µL of ice-cold 1x extraction buffer.

  • Incubate at least for Duration00:15:00 at Temperature4 °C
  • Centrifuge Centrifigation15000 rpm, 00:10:00 Temperature4 °C
  • Transfer the supernatant to a new tube. Store the samples at Temperature-80 °C until further processing.
25m
Quality Control
  • Add loading dye to the sample before running it on SDS-PAGE. Load 1% of the whole brain lysate and 10% of LysoIP.

  • Detect lysosomal markers (LAMP1, Cathepsin B), Golgi marker (Golgin-97), endoplasmic reticulum marker (Calreticulin), and mitochondrial marker (VDAC) to confirm the enrichment of lysosomes.