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Subcellular Protein Fractionation Kit for Cultured Cells


Segregate and enrich proteins from five cellular compartments.

Subcellular Protein Fractionation Kit for Cultured Cells

The Thermo Scientific Subcellular Protein Fractionation Kit for Cultured Cells enables segregation and enrichment of proteins from five different cellular compartments.

The kit enables the stepwise separation and extraction of cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal proteins from mammalian cultured cells. Extracts obtained with the Subcellular Protein Fractionation Kit for Cultured Cells are compatible with a variety of downstream applications including Western blotting, protein assays, electrophoretic mobility shift assays and reporter-gene and enzyme-activity assays.

Fractionation of subcellular proteins enables protein localization assessment and protein enrichment from specific cellular compartments. The Thermo Scientific Subcellular Protein Fractionation Kit includes a combination of reagents for stepwise lysis of cells into functional cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein fractions in less than 3 hours. Extracts from each subcellular compartment generally have less than 15% contamination between fractions, which is sufficient purity for most experiments studying protein localization and redistribution.

Highlights:

  • Concise – extract functional cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein fractions in less than 3 hours
  • Convenient – perform a simple procedure without using ultracentrifugation over gradients
  • Complete – obtain cytoplasmic, membrane, soluble nuclear, chromatin-bound and cytoskeletal protein fractions from a single kit
  • Cultured Cells – developed and optimized for use with cultured mammalian cells; for tissue samples use the Subcellular Fractionation Kit for Tissues
  • Compatible – use extracts for downstream applications such as protein assays, Western blotting, gel-shift assays and enzyme activity assays

Applications for subcellular fractionation:

  • Extract and enrich proteins from different cellular compartments
  • Isolate cytoplasmic, nuclear, membrane and cytoskeletal proteins from the same cells
  • Profile cells for proteins from the cytoplasm, nucleus, membrane and cytoskeleton
  • Study protein translocation

Product Details:

The Subcellular Protein Fractionation Kit for Cultured Cells contains four extraction buffers, a stabilized nuclease and Thermo Scientific Halt Protease Inhibitor Cocktail. Each kit has enough reagents to fractionate 50 cell pellets of 20µL each, equivalent to approximately 2g of cell paste. (For tissue samples, use our Subcellular Protein Fractionation Kit for Tissue.)

The first kit reagent, when added to a cell pellet, causes selective membrane permeabilization, releasing soluble cytoplasmic contents. The second reagent dissolves plasma, mitochondria and ER-golgi membranes but does not solubilize the nuclear membranes. After recovering intact nuclei by centrifugation, a third reagent yields the soluble nuclear extract. An additional nuclear extraction with micrococcal nuclease is performed to release chromatin-bound nuclear proteins. The recovered insoluble pellet is then extracted with the final reagent to isolate cytoskeletal proteins.

Protocol summary for the Thermo Scientific Subcellular Protein Fractionation Kit
Schematic overview of the subcellular fractionation procedure. Cellular compartments are sequentially extracted by incubating cells with cytoplasmic extraction buffer (CEB) followed by membrane extraction buffer (MEB) and nuclear extraction buffer (NEB). Adding micrococcal nuclease (MNase) to NEB extracts chromatin-bound proteins from the cell pellet before adding the pellet extraction buffer (PEB) to solubilize cytoskeletal proteins.

 

 Cytoplasmic   Cytoplasmic HSP90 Western Data HSP90 Western blots of fractionated cellular proteins. HeLa cells (2 x 10^6) were fractionated using the Subcellular Protein Fractionation Kit. Normalized portions of each extract (10µg) were analyzed by Western blotting using specific antibodies against proteins from various cellular compartments including cytoplasmic (HSP90), plasma membrane (EGFR), endoplasmic reticulum (calreticulin), nuclear soluble (SP1 and HDAC2); chromatin-bound (histone 3); and cytoskeleton (cytokeratin 18 and vimentin). The blots were probed with goat anti-rabbit (H+L) HRP or goat anti-mouse (H+L) HRP and detected with Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Part No. 34076). The results demonstrate clear fractionation of each protein with low cross contamination among fractions.

CE: cytoplasmic extract,
ME: membrane extract,
NE: nuclear extract,
CB: chromatin-bound extract,
PE: pellet extract.
 Membrane Membrane EGFR Western Data EGFR
Membrane calrecticulin Western Data calreticulin   
 Nuclear
 Soluble
Nuclear Soluble SP1 Western Data SP1
Nuclear Soluble HDAC2 Western Data HDAC2
 Chromatin
 Bound
  Chromatin Bound histone 3 Western Data histone 3
 Cytoskeletal Cytoskeletal vimentin Western Data vimentin
Cytoskeletal cytokeratin 18 Western Data cytokeratin
18
    CE ME NE CP PE    

 

Western blot showing translocation of NFkB and PKCa
Western blot analysis of protein translocation. Panel A. A549 cells (2 x 10^6) were either mock-treated (-) or incubated (+) with 20µg/mL TNFα for 20 minutes and fractionated using the Subcellular Protein Fractionation Kit. Each extract (10µg) was analyzed by Western blot using an anti-NFκB p65 antibody. Panel B. Serum-starved HeLa cells (2 x 10^6) were either mock-treated (-) or incubated (+) with 1µM PMA for 20 minutes and fractionated. Each extract (10µg) was analyzed by Western blot using an anti-PKCα antibody. Goat anti-rabbit or anti-mouse (H+L) HRP was used as the secondary antibody, and SuperSignal West Dura Chemiluminescent Substrate (Part No. 34076) was used for detection.

References:

  1. Cascio S. et al. (2011) MUC1 protein expression in tumor cells regulates transcription of proinflammatory cytokines by forming a complex with nuclear factor-kappaB p65 and binding to cytokine promoters: importance of extracellular domain. J Biol Chem. 286, 42248-56.
  2. Costello D. A. et al. (2011) Long term potentiation is impaired in membrane glycoprotein CD200-deficient mice: A role for toll-like receptor activation. J Biol Chem. 286, 34722-32.
  3. Draker R. et al. (2011) USP10 deubiquitylates the histone variant H2A.Z and both are required for androgen receptor-mediated gene activation. Nucleic Acids Res. 39, 3529-42.
  4. Liao A. et al. (2011) Therapeutic efficacy of FTY720 in a rat model of NK-cell leukemia. Blood. 118, 2793-800.
  5. Loo L. H. et al. (2009) Heterogeneity in the physiological states and pharmacological responses of differentiating 3T3-L1 preadipocytes. J Cell Biol. 187, 375-84.
  6. Richard D. J. et al. (2011) hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex. Nucleic Acids Res. 39, 1692-702.
  7. Roger L. et al. (2010) Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells. J Cell Sci. 123, 1295-305.
  8. Roy G. et al. (2012) ApoB-100-containing lipoproteins are major carriers of 3-iodothyronamine in circulation. J Biol Chem. 287, 1790-800.

Related Resources:

Review of cell fractionation and organelle isolation

Related Products:

Subcellular Protein Fractionation Kit for Tissue
Mem-PER Plus Membrane Protein Extraction Kit
NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit
Organelle Isolation Kits
Protease Inhibitors and Phosphatase Inhibitors


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
78840 Subcellular Protein Fractionation Kit for Cultured Cells
Formulation: Multi-component kit
Sufficient For: 50 samples each containing 2 million cells (20µL packed)

Kit Contents:
Cytoplasmic Extraction Buffer, 10mL
Membrane Extraction Buffer, 10mL
Nuclear Extraction Buffer, 10mL
Pellet Extraction Buffer, 5mL
Micrococcal Nuclease, 150µL
Calcium Chloride, 100mM, 250µL
Halt Protease Inhibitor Cocktail (100X), 350µL
35mL kit Product Instructions for product #78840 Subcellular Protein Fractionation Kit for Cultured Cells MSDS for product #78840 Subcellular Protein Fractionation Kit for Cultured Cells Certificate of Analysis for product #78840 Subcellular Protein Fractionation Kit for Cultured Cells $400.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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