Jul 07, 2023

Public workspaceNuclear cytoplasmic fractionation

DOI
dx.doi.org/10.17504/protocols.io.ewov1oejylr2/v1
  • Narayana Yadavalli1,2,3,4,5,
  • Shawn M. Ferguson1,2,3,4,6,5
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Berrak Ugur
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.ewov1oejylr2/v1
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. Nuclear cytoplasmic fractionation. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1oejylr2/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: May 24, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82369
Keywords: nuclear cytoplasmic fractionation, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes nuclear cytoplasmic fractionation.
Attachments
Icon for 724-1816.docx
724-1816.docx
15KB
Materials
Regents required

  • PBS
  • SDS
  • Benzonase
  • Hypotonic buffer
  • 10 mM Hepes (pH 7.9)
  • 10 mM KCl
  • 0.1 mM EDTA
  • 0.1 mM EGTA
  • 1 mM dithiothreitol (DTT)
  • high-salt buffer
  • 20 mM HEPES
  • 400 mM NaCl
  • 1 mM EDTA
  • 1 mM EGTA
  • 1 mM DTT
  • 0.5% NP-40
Nuclear cytoplasmic fractionation
Nuclear cytoplasmic fractionation
6h 5m
6h 5m
Treat 500,000 BMDM’s with DMSO or Concentration50 nanomolar (nM) MLi-2 for Duration06:00:00 .
6h
After the treatment, wash the cells 3X in PBS.
Wash
Harvest the cells in Amount500 µL of ice-cold hypotonic buffer.
Homogenize the cells with 20 strokes of a Dounce homogenizer.
To Amount100 µL of this homogenate, add SDS (1% final) and Amount25 U of Benzonase (Novagen). Use this as total lysate.
Pipetting
Spin the rest of the homogenate at Centrifigation14000 rpm, 4°C, 00:05:00 .

5m
Centrifigation
Collect the supernatant (cytoplasmic fraction) into a new tube.
Resuspend the pellet (nuclear fraction) in Amount200 µL of high-salt buffer and solubilize with SDS (1% final) in the presence of Amount25 U of Benzonase.
Measure protein concentration with the BCA reagent (Thermo Scientific), and subsequently analyze the samples by immunoblotting.
Analyze