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Subcellular localization is crucial for the proper functioning of a protein. Deregulation of subcellular localization may lead to pathological consequences and result in diseases like cancer. Immuno-fluorescent staining and subcellular fractionation can be used to determine localization of a protein. Here we discuss a protocol to separate the nuclear, cytosolic, and membrane fractions of cultured human cell lines using a centrifuge and ultracentrifuge. The membrane fraction contains plasma membranes and ER-golgi membranes, but no mitochondria or nuclear structures. The fractions can be further analyzed using Western blotting. This protocol is based on that from Dr. Richard Patten at Abcam, and was modified and utilized in a publication by Huang et al. (2012).

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Subcellular Fractionation of Cultured Human Cell Lines

Cancer Biology > General technique > Biochemical assays > Protein analysis
Authors: Zhuoyou Yu
Zhuoyou YuAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
Bio-protocol author page: a597
Zhiguang Huang
Zhiguang HuangAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
Bio-protocol author page: a598
 and Maria Li Lung
Maria Li LungAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
For correspondence: mlilung@hku.hk
Bio-protocol author page: a101
Vol 3, Iss 9, 5/5/2013, 15207 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.754

[Abstract] Subcellular localization is crucial for the proper functioning of a protein. Deregulation of subcellular localization may lead to pathological consequences and result in diseases like cancer. Immuno-fluorescent staining and subcellular fractionation can be used to determine localization of a protein. Here we discuss a protocol to separate the nuclear, cytosolic, and membrane fractions of cultured human cell lines using a centrifuge and ultracentrifuge. The membrane fraction contains plasma membranes and ER-golgi membranes, but no mitochondria or nuclear structures. The fractions can be further analyzed using Western blotting. This protocol is based on that from Dr. Richard Patten at Abcam, and was modified and utilized in a publication by Huang et al. (2012).

Keywords: Nuclear, Cytosolic, Membrane, Centrifugation, Fractionation

Materials and Reagents

  1. Sucrose
  2. HEPES
  3. Potassium chloride (KCl)
  4. Magnesium chloride (MgCl2)
  5. Ethylene diamine tetraacetic acid (EDTA)
  6. Ethylene glycol tetraacetic acid (EGTA)
  7. Dithiothreitol (DTT)
  8. Tris (Affymetrix-USB, catalog number: 75825)
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 13565)
  10. Nonidet P40 substitute (NP40)
  11. Sodium deoxycholate
  12. Glycerol
  13. Sodium dodecyl sulfate (SDS)
  14. Protease inhibitor (PI) cocktails (F. Hoffmann-La Roche, catalog number: 11836145001)
  15. Methanol
  16. Acetic acid
  17. Brilliant Blue R (Affymetrix, catalog number: 32826)
  18. Phosphate buffered saline (PBS)
  19. Histone H3 antibody (Cell Signaling Technology, catalog number: 9715)
  20. Alpha-tubulin antibody (GeneTex, catalog number: GTX108784)
  21. Cell scraper (BD Biosciences, Falcon®, catalog number: 353086)
  22. Subcellular fractionation buffer (SF buffer) (see Recipes)
  23. Nuclear Lysis buffer (NL buffer) (see Recipes)
  24. Brilliant Blue R staining solution and destaining solution (see Recipes)

Equipment

  1. 4 °C Microcentrifuge (Eppendorf, catalog number: 5415R);
  2. Ultracentrifuge (Beckman Coulter, model number: Optima TLX)
  3. Optional: Sonicator (Sonics, model number: VC505)
  4. Whatman filter paper
  5. 37 °C incubator
  6. 1.5 ml Eppendorf microtubes
  7. Tube roller (Maplelab-scientific, model number: MTR-1D)

Procedure

Note: Keep the sample at 4°C on ice at ALL times! All buffers must be ice-cold when used. All centrifugations are done in the Eppendorf Microcentrifuge unless stated otherwise.

  1. Culture cells on 100 mm culture plate until 75% confluent in a 37 °C incubator supplied with 5% CO2. For beginners, the well-studied HEK293 and its derivatives are recommended for easy maintenance and ectopic protein expression.
    Note: This is for adherent cell. Suspension cells may need centrifugation before lysis.
  2. Wash twice with ice-cold PBS and immediately add 500 μl per 100 mm plate of SF buffer and put on ice, use cell scraper to collect lysate and transfer to a 1.5 ml Eppendorf tube. If multiple samples are collected, process one specimen at a time.
  3. Agitate the lysates at 4 °C for 30 min at around 30-50 rpm on the tube roller.
  4. Centrifuge at 720 x g at 4 °C for 5 min. Carefully transfer the supernatant to a new 1.5 ml tube for future use. Keep the pellet for next step.
  5. Wash the pellet with 500 μl of SF buffer and disperse the pellet with a pipette.
  6. Centrifuge the pellet at 720 x g at 4 °C for 10 min.
  7. Remove the supernatant and resuspend the pellet in NL buffer. Agitate and incubate at 4 °C for 15 min.
    Optional: Sonicate the pellet on ice (2 x 3 sec sonication, separated by 3 sec resting, under 30% full amplitude power. On ice!). This is the nuclear fraction including nuclear membranes.
  8. Centrifuge the supernatant from step 4 at 10,000 x g at 4 °C for 10 min.
  9. Carefully transfer the supernatant to a new 1.5 ml tube. This is the cytosolic and membrane fraction.
  10. Centrifuge the cytosolic and membrane fraction from step 9 in an ultracentrifuge. Ultracentrifuge at 100,000 x g at 4 °C for 1 h. Carefully transfer the supernatant to a new 1.5 ml tube. This is the cytosolic fraction. 
  11. Wash the pellet with 500 μl of SF buffer and re-suspend by pipetting.
  12. Ultracentrifuge the pellet at 100,000 x g at 4 °C for 1 h.
  13. Remove the supernatant and re-suspend the pellet in NL buffer.
    Optional: Sonicate the pellet on ice (same setting as for nuclear fraction in step 7). This is the membrane fraction.
  14. Internal loading control for Western blotting could be used to make sure each fraction does not cross-contaminate others; but relative amount can also be determined between samples to ensure equal loading. For example, alpha-tubulin is used for the cytosolic fraction; histone H3 is used for the nuclear fraction; Brilliant Blue R is used for staining for the membrane fraction. Use an extra gel for the loading controls. Alpha-tubulin and histone H3 are probed after protein is transferred onto a PVDF membrane. Brilliant Blue R staining can be applied directly to the SDS-PAGE gel (Figure 1).
    Note: Brilliant Blue R is used here for monitoring relative amount of protein loading across different samples, but is unable to show cross contamination. Membrane proteins such as EGF receptor and integrins may be used to confirm cross-contamination between the membrane and other fractions. However the recycling of these membrane proteins can be an issue, which may appear to be false-positive cross-contamination.


    Figure 1. Western blotting of nuclear, cytoplasmic, and membrane fractions with internal controls. Alpha-tubulin and histone H3 are used for the cytoplasmic and nuclear fractions, respectively; Brilliant Blue R staining is applied for the membrane fraction.

Recipes

  1. Subcellular fractionation buffer (SF buffer)

    Stocks                    
    50 ml 1x solution      
    250 mM Sucrose
    -
    4.28 g
    20 mM HEPES (pH 7.4)
    1 M
    1 ml
    10 mM KCl
    -
    0.0373 g
    1.5 mM MgCl2
    1 M
    75 μl
    1 mM EDTA
    0.5 M
    100 μl
    1 mM EGTA
    0.5 M
    100 μl
  2. At time of use, add the following into 10 ml of SF buffer
                                                   
    Stocks
    10 ml 1x solution                 
    1 mM DTT
    1 M
    10 μl
    PI cocktail
    40x (dissolve 1 tablet in 2 ml dd H2O)
    250 μl
  3. Nuclear Lysis buffer (NL buffer)

    Stocks                        
    50 ml 1x solution          
    50 mM Tris HCl (pH 8)
    1 M
    2.5 ml
    150 mM NaCl
    1 M
    7.5 ml
    1% NP-40
    20%
    2.5 ml
    0.5% sodium deoxycholate
    10%
    2.5 ml
    0.1% SDS
    10%
    0.5 ml
  4. At time of use, add the following into 10 ml of NL buffer
                               
    Stocks              
    10 ml 1x solution
    PI cocktail
    40x
    250 μl
    10% glycerol
    -
    1 ml

    Note: DTT can be added if further delicate experiments such as IP and ChIP are needed; current recipe without DTT is fine for Western blotting.
  5. Brilliant Blue R staining solution and destaining solution
    For staining solution, dissolve 1 g of Brilliant Blue powder in 1 L of 50% Methanol/10% AceticAcid/40% H2O (all [v/v]) solution. Stir until dissolved and (optional) filter through Whatman filter paper.
    For destaining, make a 10% AceticAcid/15% Methanol/75% H2O solution.
    Procedures: Place the gel containing the proteins of interest in a plastic container and cover with fresh staining solution. Shake it for 1 h at room temperature (or overnight). Remove the staining solution and add destaining solution. Put three sheets of fine-grade tissue paper in the container. Shake it until gel is fully destained. Constantly replace the solution and tissue paper.

Acknowledgments

This protocol is based on that from Dr. Richard Patten at Abcam (see Reference 1), and was modified and utilized in a publication by Huang et al. (2012).

References

  1. Patten R. Procedure for separating nuclear, membrane, and cytoplasmic cell fractions using centrifugation methods.
  2. Huang, Z., Cheng, Y., Chiu, P. M., Cheung, F. M., Nicholls, J. M., Kwong, D. L., Lee, A. W., Zabarovsky, E. R., Stanbridge, E. J., Lung, H. L. and Lung, M. L. (2012). Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties. Oncogene 31(32): 3709-3720.


How to cite: Yu, Z., Huang, Z. and Lung, M. L. (2013). Subcellular Fractionation of Cultured Human Cell Lines. Bio-protocol 3(9): e754. DOI: 10.21769/BioProtoc.754; Full Text



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3/18/2015 12:29:15 PM  

Angela Russo
UIC

Hello,
I would like to know the pH of EDTA and EGTA stocks for the preparation of the fractionation buffer.
Thanks,
Angela

3/27/2015 2:07:45 AM  

Maria Lung (Author)
Clinical Oncology Department,The Univerisity of Hong Kong

Dear Angela,

we use 0.5M pH8 (adjusted using NaOH) EDTA and EGTA stocks for the preparation.

yours
Valen Z. YU

Reply

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