Apr 23, 2026

LysoIP from iPSC-derived mDANs

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx4qpol8j/v1
  • 1National Human Genome Research Institute
Yu Chen
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx4qpol8j/v1
Protocol Citation: Yu Chen, Chase Chen 2026. LysoIP from iPSC-derived mDANs. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx4qpol8j/v1
Manuscript citation:
Bidirectional regulation of glycoprotein nonmetastatic melanoma protein B by β-glucocerebrosidase deficiency in GBA1 isogenic dopaminergic neurons from a patient with Gaucher disease and parkinsonism
Chase Chen, Charis Ma, Richard Sam, Jens Lichtenberg, Tiffany Chen, Ying Hao, Ziyi Li, Isabelle Kowal, Kate Andersh, Yue Andy Qi, Gani Perez, Ellen Hertz, Yan Li, Darian Williams, Mark J. Henderson, Morgan Park, Xuntian Jiang, Pilar Alvarez Jerez, Cornelis Blauwendraat, Ellen Sidransky, Yu Chen. doi: https://doi.org/10.1101/2025.06.23.661126
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 15, 2025
Last Modified: April 23, 2026
Protocol  Integer ID: 229908
Keywords: ASAPCRN, LysoIP, mDAN, iPSC, lysosomes from ipsc, lysosome, mdans this protocol, derived mdan, mdan
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
This protocol details the steps to pull down lysosomes from iPSC-derived mDANs.
Materials
Anti-HA Magnetic Beads, 5mL, supplied at 10mg/mL (Pierce, 88837)
Protease and Phosphatase Mini Tablets EDTA free (Pierce, A32959)
KPBS Buffer (136 mM KCl, 10 mM KH2PO4, pH 7.25 adjusted with KOH) – HPLC grade chemicals
Lysis Buffer (2% SDS + 50 mM TEAB pH 8.5), diluted from UltraPure SDS Solution, 10%, ThermoFisher Scientific, 15553027, and Triethylammonium bicarbonate buffer, 1.0 M, pH 8.5±0.1, Millipore Sigma, T7408.  
DynaMag Magnet (ThermoFisher Scientific, 12321D)
Motorized Tissue Homogenizer (TH) (Omni, TH115) + 7 mm Soft Tissue Omni Tip‱ Plastic Homogenizing Probes (SKU: 30750)
Pre-wash beads according to manufacturer’s protocol
Premake KPBS buffer with protease + phosphatase inhibitors not more than 1 hr before IP
Aliquot 150 uL anti-HA Beads in 1.5 mL tubes.
Wash 3 times with 500 uL and 300 uL of KPBS + protease/phosphatase inhibitors to remove NaN3 and Tween-20.
Leave beads in 300 uL of KPBS + protease/phosphatase inhibitors until cell homogenates are ready.
mDAN Collection
Start with 35 x 106 iPSC-derived mDANs in a T-175 flask for each technical replicate of Lyso IP
Wash cells with ice cold DPBS once and aspirate DPBS
Scrape mDANs off in 10 mL ice cold KPBS buffer and spin down 1000 x g, 4°C

Aspirate KPBS and resuspend the pelleted mDANs in 950 uL of KPBS + protease/phosphatase inhibitors and save 25 uL for Western blot as “input”
mDAN Homogenization
Homogenize for 3-5 rounds. In each round, run homogenizor for 00:00:10 at max power for and wait for bubbles to disappear before proceeding to the next round.

For best efficiency use 15 mL conical tube (not 50 mL) and leave samples on ice while homogenizing.
Lysosome Pulldown
Centrifuge homogenates at 1000 x g, 4°C for 2 min to pellet nuclei. The supernatant contains cellular organelles including lysosomes.

Note: Supernatant should be cloudy and yellowish if protein concentration is high.
Mix pre-washed anti-HA beads in KPBS + inhib with cell homogenates for 00:05:00 on rotor at 4 °C .

Wash anti-HA beads with bound organelles 3 times with 1 mL KPBS + Protease/Phosphatase inhibitors.
Protein Elution from anti-HA Beads for Proteomics
Add 100 µL of Lysis Buffer (2% SDS, 50 mM TEAB pH 8.5) to each technical replicate of LysoIP samples.

Boil samples for 00:10:00 at 100 °C

Let samples cool down to room temperature.
Settle anti-HA beads using magnetic stand and transfer supernatant to a new tube.
Continue with S-trap protocol to prepare for proteomics analysis.
Protocol references
1.         Abu-Remaileh, M., et al., Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes. Science, 2017. 358(6364): p. 807-813.
2.         Wyant, G.A., et al., mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient. Cell, 2017. 171(3): p. 642-654 e12.
3.         Lim, C.Y., et al., ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann-Pick type C. Nat Cell Biol, 2019. 21(10): p. 1206-1218.
4.         Castellano, B.M., et al., Lysosomal cholesterol activates mTORC1 via an SLC38A9-Niemann-Pick C1 signaling complex. Science, 2017. 355(6331): p. 1306-1311.
5.         Davis, O.B., et al., NPC1-mTORC1 Signaling Couples Cholesterol Sensing to Organelle Homeostasis and Is a Targetable Pathway in Niemann-Pick Type C. Dev Cell, 2021. 56(3): p. 260-276 e7.
6.         Xiong, J., et al., Rapid affinity purification of intracellular organelles using a twin strep tag. J Cell Sci, 2019. 132(24).
7.         Wyant, G.A., et al., NUFIP1 is a ribosome receptor for starvation-induced ribophagy. Science, 2018. 360(6390): p. 751-758.