Oct 14, 2024

Public workspaceSDS-PAGE and immunoblotting to assess whole-cell lysates and Endo-IPs and Lyso-IPs in hESCs and iNeurons

DOI
https://dx.doi.org/10.17504/protocols.io.q26g71eq9gwz/v1
  • Frances V Hundley1,2,
  • Miguel A. Gonzalez-Lozano1,2,
  • Harper JW1,2
  • 1Harvard Medical School;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Frances V Hundley
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g71eq9gwz/v1
External link: https://doi.org/10.1038/s41586-025-09059-y
Protocol Citation: Frances V Hundley, Miguel A. Gonzalez-Lozano, Harper JW 2024. SDS-PAGE and immunoblotting to assess whole-cell lysates and Endo-IPs and Lyso-IPs in hESCs and iNeurons. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71eq9gwz/v1
Manuscript citation:
Gonzalez-Lozano, M.A., Schmid, E.W., Miguel Whelan, E. et al. EndoMAP.v1 charts the structural landscape of human early endosome complexes. Nature 643, 252–261 (2025). https://doi.org/10.1038/s41586-025-09059-y
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 02, 2024
Last Modified: October 14, 2024
Protocol Integer ID: 108855
Keywords: ASAPCRN, negative control markers from other organelle, endolysosomal protein, cell lysate, other organelle, negative control marker, ineuron, ips in hesc, protein, ips from hesc, general protocol for western blotting
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-025160
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Abstract
We present a general protocol for Western blotting to detect endolysosomal proteins and negative control markers from other organelles to assess whole-cell lysates and Endo-IPs and Lyso-IPs from hESCs and iNeurons.

Troubleshooting
SDS-PAGE and immunoblotting
5m
For whole-cell lysates, prepare samples following standard protocols, and final samples should be in LDS buffer with DTT or similar. For Endo-IP and/or Lyso-IP from hESCs and/or iNeurons, post-nuclear supernatant (PNS), flow through, and eluate samples should be in LDS buffer with DTT or similar. Incubate samples at Temperature80 °C for Duration00:05:00 .

5m
Load samples into a 4-20% Criterion TGX Stain-free precast gel (Bio-Rad) and separate by electrophoresis in 1xTris/Glycine/SDS buffer.
Scan gel for total protein using a ChemiDoc MP imager (Bio-Rad).
Transfer proteins to PVDF or nitrocellulose membranes by standard wet transfer in 20% methanol Tis/Glycine buffer.
Block membrane in blocking buffer (5% non-fat dry milk or 3% BSA in TBST) at TemperatureRoom temperature for Duration01:00:00 .

1h
Incubate membrane in primary antibody solution (blocking solution plus primary antibody at 1:500-1:1,000, depending on the primary antibody) at Temperature4 °C for Duration12:00:00 toDuration16:00:00 .

1d 4h
Wash membrane six times with TBST for Duration00:05:00 each wash.

5m
Incubate membrane in secondary antibody solution (blocking solution plus secondary antibody conjugated to HRP at 1:5,000-1:10,000) at TemperatureRoom temperature for Duration01:00:00 .
1h
Wash membrane four times with TBST for Duration00:05:00 each wash.
5m
Apply Western Lightning Plus Chemiluminescence substrate (Revvity) to membrane and acquire blot images using a ChemiDoc MP imager.
Process raw image files with Image Lab software (Bio-Rad).
Protocol references
Frances V. Hundley, Miguel A. Gonzalez-Lozano, Lena M. Gottschalk, Aslan N. K. Cook, Jiuchun Zhang, Joao A. Paulo, J. Wade Harper. Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons
bioRxiv 2024.09.24.614704; doi: https://doi.org/10.1101/2024.09.24.614704