Oct 23, 2025
Sequential Detergent and Salt-based Subcellular Fractionation of Human Neural Stem Cells
- Katja Piltti1,
- Francisca Benavente-Perez1
- 1University of California, Irvine
Protocol Citation: Katja Piltti, Francisca Benavente-Perez 2025. Sequential Detergent and Salt-based Subcellular Fractionation of Human Neural Stem Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n9o8lwz/v1
Manuscript citation:
Piltti KM et al. C1q drives neural stem cell quiescence by regulating cell cycle and metabolism through BAI1. Nature Communications (In press)
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 21, 2025
Last Modified: October 23, 2025
Protocol Integer ID: 230355
Keywords: Subcellular fractionation, Sequential protein extraction, Protein enrichment, Neural stem cells, Stem cell biology, Fraction validation, Detergent-based extraction, Salt-based extraction, Cytoplasmic fraction, Membrane protein fraction, Nuclear protein fraction, Insoluble protein fraction, human neural stem cells sequential fractionation, human neural stem cell, based subcellular fractionation, neural stem cell, intracellular localization of p32, nuclear protein fractions for validation, nuclear protein fraction, showing intracellular localization, cell,
Funders Acknowledgements:
Wings for Life
Grant ID: WFL-US-01/18
Abstract
Sequential fractionation of neural stem cells was performed to obtain cytoplasmic, membrane, and nuclear protein fractions for validation of Imagestream, immunocytochemistry, and biotin-enrichment data showing intracellular localization of p32 in human neural stem cells.
Guidelines
14. Centrifuge again at 12,000 rcf for 15 minutes.
15. Collect the supernatant as the TX-100+ KCl-nuclear protein fraction.
Insoluble Protein Fraction:
16. Resuspend the final pellet in 20μL of 5X SDS-PAGE sample loading buffer (250mM Tris-HCl, pH 6.8, 10% SDS, 30% glycerol, 10mM DTT, 0.05% bromophenol blue).
17. Boil the sample for 5 minutes to denature proteins, preparing them for analysis.
Analysis:
18. Analyze 10μg of protein from each fraction using Western blot employing antibodies specific to compartment markers, and loading controls.
Materials
- Cold Dulbecco's Phosphate-Buffered Saline (DPBS without Ca2+/Mg2+)
- Cell scraper
- PBS (0.1 mM Ca²⁺ and 1 mM Mg²⁺)
- Protease inhibitor (Roche Applied Science, cOmplete ULTRA, 05892970001)
- Phosphatase inhibitor (Roche Applied Science, PhosSTOP, 04906837001)
- 1 mL syringes
- 27.5 G needle
- 35Ga needle
- Lysis buffer (50mM Tris-HCl, pH 7.5, 100mM NaCl, 0.5% Triton X-100)
- 0.5M KCl
- 5X SDS-PAGE sample loading buffer (250mM Tris-HCl, pH 6.8, 10% SDS, 30% glycerol, 10mM DTT, 0.05% bromophenol blue)
Troubleshooting
Cell Detachment and Initial Preparation
Culture hNSC on poly-L-ornithine and laminin-coated 6-well plates in growth medium (GM).
Wash hNSC twice with cold Dulbecco's Phosphate-Buffered Saline without Ca2+/Mg2+ (DPBS -/-).
Detach the cells using a cell scraper in 500 μL PBS (0.1 mM Ca²⁺ and 1 mM Mg²⁺) supplemented with protease inhibitor (Roche Applied Science, cOmplete ULTRA, 05892970001) and phosphatase inhibitor (Roche Applied Science, PhosSTOP, 04906837001). Keep on ice to prevent protein degradation.
Mechanical Disruption
Homogenize the detached cells by passing them through a 1 mL syringe fitted with a 27.5 G needle, using 10 up-and-down strokes. This disrupts the plasma membrane, releasing cytoplasmic contents.
Cytoplasmic Fraction Collection
Incubate the homogenate on ice for 10 minutes to ensure complete lysis.
Centrifuge at 12,000 g for 15 minutes at 4°C. Ensure the centrifuge is pre-cooled.
Carefully collect the supernatant as the PBS-soluble cytoplasmic protein fraction.
Membrane/Structural Protein Fraction
Resuspend the remaining cell pellet in 500μL of lysis buffer containing 50mM Tris-HCl (pH 7.5), 100mM NaCl, 0.5% Triton X-100, and protease and phosphatase inhibitors. The Triton X-100 helps solubilize membrane proteins.
Use a 1 mL syringe fitted with a 35 G needle for further disruption, performing 10 additional up-and-down strokes.
Incubate on ice for 10 minutes followed by centrifugation at 12,000 g for 15 minutes at 4°C.
Collect the supernatant as the detergent (TX-100)-soluble membrane/subcellular structure protein fraction.
Nuclear and Caveolae Protein Fraction
Resuspend the remaining pellet in 200μL of lysis buffer with 0.5M KCl and protease and phosphatase inhibitors to extract nuclear proteins.
Incubate on ice for 10 minutes to allow salt to permeabilize the nuclear envelope.
Centrifuge again at 12,000 g for 15 minutes.
Collect the supernatant as the TX-100+ KCl-nuclear protein fraction.
Insoluble Protein Fraction
Resuspend the final pellet in 20μL of 5X SDS-PAGE sample loading buffer (250mM Tris-HCl, pH 6.8, 10% SDS, 30% glycerol, 10mM DTT, 0.05% bromophenol blue).
Boil the sample for 5 minutes to denature proteins, preparing them for analysis.
Analysis
Analyze 10μg of protein from each fraction using Western blot employing antibodies specific to compartment markers, and loading controls.