Feb 11, 2026

Public workspaceRapid Isolation of Lysosomes (LysoIP) from iPSC-Derived Neurons for Proteomics, with Options for Lipidomics/Metabolomics

DOI
https://dx.doi.org/10.17504/protocols.io.x54v9bw9ql3e/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.x54v9bw9ql3e/v1
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Rapid Isolation of Lysosomes (LysoIP) from iPSC-Derived Neurons for Proteomics, with Options for Lipidomics/Metabolomics. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9bw9ql3e/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: February 11, 2026
Last Modified: February 11, 2026
Protocol Integer ID: 243070
Keywords: rapid immunopurification of lysosome, rapid isolation of lysosome, metabolomics this protocol, proteomic, derived neurons for proteomic, metabolomic, lysosome, extraction compatible with downstream mass spectrometry, rapid immunopurification, options for lipidomic, cultured ineuron, downstream mass spectrometry, multiple dishes for lipidomic, lysosomal ha tag, lipidomic, lysoip, ineuron
Abstract
This protocol describes rapid immunopurification of lysosomes (LysoIP) from cultured iNeurons expressing a lysosomal HA tag (e.g., TMEM192-3xHA). The workflow is optimized for proteome analysis from one 10-cm dish (~8–10×10^6 iNeurons) and can be scaled by pooling multiple dishes for lipidomics/metabolomics. The method relies on cold, rapid handling; gentle homogenization; anti-HA magnetic capture; stringent washes; and extraction compatible with downstream mass spectrometry.  
Materials

Reagents


  • Anti-HA magnetic beads (e.g., Thermo Fisher; same class as used in template)  
  • PBS, pH 7.4 (ice-cold)  
  • Complete EDTA-free Protease Inhibitor Cocktail (Roche)  
  • Optima LC/MS water (if doing metabolite/lipid workflows)  
  • KPBS (for metabolite/lipid-compatible washes): 136 mM KCl, 10 mM KH2PO4, pH 7.25 (KOH; in MS-grade water)  
  • Optima LC/MS methanol (for metabolite extraction)  
  • (Optional) isotopically labeled internal standards for metabolomics  


Buffers


A) Ice-cold PBS + Protease inhibitors (PBS+PI)

  • PBS pH 7.4 + Complete EDTA-free PI (fresh)

B) 1× Extraction Buffer (for protein extraction)

  • 50 mM HEPES pH 7.4
  • 40 mM NaCl
  • 2 mM EDTA
  • 1% Triton X-100
  • 1.5 mM sodium orthovanadate
  • 30 mM sodium fluoride
  • 10 mM sodium pyrophosphate
  • 10 mM sodium β-glycerophosphate
  • Complete EDTA-free PI  

C) 80% MeOH (v/v), pre-chilled to freezing

  • 80% methanol in LC/MS water (add internal standards if desired)(For lipidomics or metabolomics)

Equipment


  • DynaMag (or equivalent) spin magnet  
  • Pre-chilled microcentrifuge (4 °C)  
  • Cell lifter/scraper
  • 1.5 mL low-bind tubes (recommended)
  • U-100 29G x 1/2 for homogenization
  • Rotator (cold room or 4 °C)

Troubleshooting
Before you start (Critical)
  • Pre-chill all buffers, tubes, magnet, and centrifuge to 4 °C. 
  • Keep everything on ice and minimize time from harvest → beads. 
  • Label tubes for each sample (example structure used in the template: post-homogenization, whole-cell/whole-homogenate, beads, final IP). 
Input guidance (iNeurons)

  • Proteomics (recommended starting point): 1 × 10-cm dish with ~8–10×10^6 differentiated cells per condition.

  • Lipidomics/metabolomics: plan to pool multiple 10-cm dishes per condition (start with ~3 dishes; exact number depends on platform sensitivity and expected lysosome yield—optimize empirically).

Preparation of anti-HA beads

  1. Pool beads: use 100 µL beads per sample.
  2. Wash beads 3X
  • For proteomics: wash 3× with PBS+PI  (See Materials)
  • For metabolomics/lipidomics: wash 3× with cold KPBS (MS-grade)  

3. Resuspend beads to the original pooled volume and aliquot 100 µL beads into a labeled 1.5 mL tube for each sample.  

iNeuron harvesting (10-cm dish)

  1. Place the plate on ice. Aspirate media.
  2. Wash 2× with ice-cold PBS (gentle).  
  3. Add ~950 µL ice-cold PBS+PI (proteomics) or KPBS (metabolomics) to the dish.  
  4. Scrape with a cell lifter and transfer suspension to a pre-chilled 2 mL tube.
  5. Spin 1,000×g, 2 min, 4 °C.  
  6. Aspirate supernatant and resuspend pellet in 950 µL ice-cold PBS+PI (or KPBS for metabolomics).  
  7. Take 25 µL as “whole cell/homogenate input” control.  
Homogenization

  1. Homogenize the remaining suspension by passing through an insulin syringe 2X (keep on ice; avoid bubbles).

2. Clarify nuclei/unbroken cells: spin 1,000×g, 2 min, 4 °C.  

3. Carefully transfer the supernatant (organelle-containing fraction) to the tube containing 100 µL washed anti-HA beads. Avoid disturbing the pellet.  

Immunoprecipitation

  1. Incubate on a gentle rotator at 4 °C for 10 min (proteomics workflow).  


  • (Metabolomics workflows often use a shorter timed 3 to 5 min capture in cold conditions; maintain consistency across samples.)  

2. Place on magnet and allow beads to collect (keep timing consistent, e.g., ~25 s).  
3. Remove supernatant.

Washes (critical for purity)

  1. Wash beads 3× with 1 mL cold PBS+PI (proteomics)  or 3× with 1 mL cold KPBS (metabolomics/lipidomics).  

  • During first wash, remove any liquid trapped in the cap; pipet up/down consistently.  

2. After the second wash, resuspend beads and transfer to a fresh tube for the third wash (clean-tube wash improves cleanliness).  

3. After final wash, remove all residual buffer.

Elution / Extraction options

Option A — Proteomics (protein extraction)

  1. Add 80 µL ice-cold 1× Extraction Buffer to beads.  

2. Incubate 15 min at 4 °C.  

3. Place on magnet, collect supernatant (protein extract), transfer to a new tube, store at −80 °C.  

4. Process the input control by adding extraction buffer 165 µL (template uses extraction buffer incubation then high-speed spin).  Spin top speed (~15,000 rpm), 10 min, 4 °C, transfer supernatant, store −80 °C.  



Option B — Polar metabolites (metabolomics)

1. Resuspend beads in 50 µL freezing-cold 80% MeOH (include internal standards if used).  
2. After final sample, magnet-separate and transfer the MeOH extract to a new tube; store −80 °C.  
3. Extract whole-cell control similarly with 80% MeOH and clear by top-speed spin (15,000 rpm, 10 min, 4 °C); store supernatant.  
Option C — Non-polar metabolites (metabolomics)
Please follow the steps below for processing nonpolar lipid samples in this protocol:
Step: Processing of nonpolar lipids samples

Protocol:
https://dx.doi.org/10.17504/protocols.io.5qpvor3dbv4o/v1