Jan 13, 2025

Public workspaceSmall-scale LysoIP

DOI
dx.doi.org/10.17504/protocols.io.yxmvmep25g3p/v1
  • 1Max Planck Institute of Biochemistry
Cole Sitron
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DOI: dx.doi.org/10.17504/protocols.io.yxmvmep25g3p/v1
Protocol Citation: Cole Sitron, Ulrich Dransfeld, F Ulrich Hartl 2025. Small-scale LysoIP. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmep25g3p/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 10, 2024
Last Modified: January 13, 2025
Protocol Integer ID: 102660
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000282
Aligning Science Across Parkinson’s
Grant ID: ASAP- 024268
Abstract
This protocol describes a technique for native immunoprecipitation of intact lysosomes (LysoIP). There are many protocols for this technique, but this protocol has been adapted to be performed at a small scale and with an elution compatible for downstream immunoblot analysis, producing just enough material for several immunoblots.
Materials
  • 10 cm plate of cells expressing TMEM192-3xHA
  • 10 cm plate of an isogenic cell line lacking this tag (to control for nonspecific binding)
  • ReagentTrypLE™ Express Enzyme (1X), phenol redThermo FisherCatalog #12605036
  • 1X PBS ReagentPBS (10X), pH 7.4Thermo Fisher ScientificCatalog #70011051
  • ReagentDMEM, high glucose, pyruvateThermo FisherCatalog #11995073 with ReagentFetal Bovine SerumGibco - Thermo FischerCatalog #10270106
  • KPBS Buffer: 10 mM KH2PO4, 136 mM KCl, pH 7.25, 1X ReagentcOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001
  • RIPA Buffer: ReagentRIPA Lysis and Extraction BufferThermo FisherCatalog #89900 , 1X cOmplete, Mini EDTA-free Protease Inhibitor Cocktail
  • ReagentNUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007 + 10% Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
  • ReagentPierce™ Anti-HA Magnetic BeadsThermo FisherCatalog #88836
  • Dounce homogenizer vessel 2 mL (VWR cat. no. 89026-386)
  • Dounce homogenizer plunger 2 mL (VWR cat. no. 89026-398)
  • Magnetic Rack
  • Centrifuge
  • Tube rotator
  • Nutator
  • ReagentPierce™ Rapid Gold BCA Protein Assay KitThermo FisherCatalog #A53225

Bead Preparation
Bead Preparation
Add Amount75 µL of anti-HA bead slurry to a tube for each sample.

Pipetting
Wash the beads 3 times with KPBS on the magnetic rack.

Wash
Resuspend the beads in Amount50 µL KPBS.

Cell Lysis
Cell Lysis
10m
10m
Aspirate the media from the cells.

Trypsinize the plate, quench with 10% FBS, and transfer to a 15 mL tube.

Spin Centrifigation300 x g, 00:03:00 .

3m
Centrifigation
Resuspend in Amount1 mL PBS and transfer to an Eppendorf tube.

Spin Centrifigation300 x g, 4°C, 00:03:00 .

3m
Centrifigation
Resuspend in Amount1 mL KPBS Buffer and transfer to a pre-cooled Douncer TemperatureOn ice .

Lyse with 30 strokes of the Douncer.

Transfer the sample to a new Eppendorf tube and clean the Douncer between each sample.

Spin Centrifigation1000 x g, 4°C, 00:04:00 .

4m
Centrifigation
Collect the post-nuclear supernatant.

Add Amount50 µL of the post-nuclear supernatant to Amount120 µL of RIPA Buffer.

Pipetting
Perform the BCA Gold assay on the RIPA Buffer-lysed post-nuclear supernatants.

Adjust each sample to Amount100 µg in Amount850 µL of KPBS.

Reserve the rest of the post-nuclear supernatant to run as an input fraction in downstream SDS-PAGE, using RIPA buffer to equalize the protein concentration between samples.

LysoIP
LysoIP
55m 30s
55m 30s
Add the Amount100 µg of sample to each tube of resuspended anti-HA beads.

Pipetting
Incubate the tube with rotation at Temperature4 °C for Duration00:20:00 .

20m
Incubation
Place the tube on the magnetic rack and remove the supernatant

Wash the beads three times with Amount1 mL KPBS, transferring the resuspended beads to a new tube on the first and third washes.

Wash
After the third wash, place the tube onto the magnetic rack and remove the supernatant.

Elute by adding Amount70 µL RIPA buffer to the tubes, gently vortexing to resuspend.

Pipetting
Place the tube on a at Temperature4 °C for Duration00:30:00 .

30m
Briefly and softly (e.g. ∼Centrifigation50 x g, 00:00:30 ) to remove any liquid that has collected in the cap.

30s
Centrifigation
Use the magnetic rack to remove the supernatant from the beads, transferring to a new tube.

The eluate and input fractions can now be denatured by adding 1/3 of the fraction volume of 4X NuPAGE LDS Sample Buffer + 10% beta-mercaptoethanol and boiling at Temperature95 °C for Duration00:05:00 .

5m
Protocol references
1. Abu-Remaileh M, Wyant GA, Kim C, Laqtom NN, Abbasi M, Chan SH, Freinkman E, Sabatini DM. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes. Science. 2017 Nov 10;358(6364):807-813. doi: 10.1126/science.aan6298. Epub 2017 Oct 26. PMID: 29074583; PMCID: PMC5704967.