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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

Published: August 12th, 2021

DOI:

10.3791/62119

1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

Subcellular fractionation is a procedure in which cells are lysed and separated into their constituent components through several methods. This technique can be used by researchers to determine protein localization in mammalian cells or for enrichment of low-abundance proteins that would otherwise be undetectable. While methods for subcellular fractionation currently exist, as do commercial kits that can be purchased, they suffer from several limitations that this procedure attempts to overcome. Most cell fractionation methods are exclusively detergent-based1,2, relying on the use of buffers containing increas....

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1. Prepare buffers and reagents

  1. Prepare fresh solutions of phosphatase and protease inhibitors.
    1. Add 17.4 mg of phenylmethanesulfonyl fluoride (PMSF)to 1 mL of 100% ethanol to prepare a 100 mM stock.
      ​NOTE: Wear protective equipment when handling PMSF as it is hazardous when ingested or inhaled and upon contact with skin or eyes. It is corrosive to eyes and skin.
    2. According to the manufacturer's instructions, prepare a commercially available protease inhibitor cocktail (.......

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A schematic flow chart of this procedure (Figure 1) visually summarizes the steps that were taken to successfully fractionate U9375 cells grown in suspension. Fractions collected from the top of the isopycnic density gradient in equal volumes (1 mL) show the purification of the mitochondrial and membrane fractions (Figure 2). Utilizing an antibody against VDAC, a protein localized to the outer mitochondrial membrane6

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This method is a modified version of a previously published approach to subcellular fractionation without the use of high-speed centrifugation11. This modified method requires more specialized equipment to achieve the best results, but is more comprehensive and consistently reproducible.

The development of the initial protocol was necessary due to an inability to separate mitochondrial and membrane samples for the analysis of protein localization during necroptosis

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This work was supported by NIH R15-HL135675-01 and NIH 2 R15-HL135675-02 to T.J.L.

....

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Name Company Catalog Number Comments
Benzonase Nuclease Sigma-Aldrich E1014
Bullet Blender Tissue Homogenizer Next Advance 61-BB50-DX
digitonin Sigma D141
end-over-end rotator ThermoFisher
Ethylenediaminetetraacetic acid (EDTA) Sigma E9884
ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) Sigma E3889
GAPDH (14C10)  Cell Signalling Technologies 2118
HEPES VWR 97064-360
Hexylene glycol Sigma 68340
Igepal Sigma I7771 Non-ionic, non-denaturing detergent
KCl Sigma P9333
Mannitol Sigma M9647
MgCl2 Sigma M8266
NaCl Sigma S9888
Na, K-ATPase a1 (D4Y7E) Cell Signalling Technologies 23565
Open-Top Polyclear Tubes, 16 x 52 mm Seton Scientific 7048
OptiPrep (Iodixanol) Density Gradient Medium Sigma D1556-250ML
phenylmethanesulfonyl fluoride (PMSF) Sigma P7626
Protease Inhibitor Cocktail, General Use VWR M221-1ML
refrigerated centrifuge ThermoFisher
S50-ST Swinging Bucket Rotor Eppendorf
Sodium dodecyl sulfate (SDS) Sigma 436143
Sodium deoxycholate Sigma D6750
sodium orthovanadate (SOV) Sigma 567540
sonicator ThermoFisher
Sorvall MX120 Plus Micro-Ultracentrifuge ThermoFisher
Stainless Steel Beads 3.2 mm Next Advance SSB32
Sucrose Sigma S0389
Tris-buffered Saline (TBS) VWR 97062-370
Tween 20 non-ionic detergent in western blotting buffers
VDAC (D73D12) Cell Signalling Technologies 4661

  1. Baghirova, S., Hughes, B. G., Hendzel, M. J., Schulz, R. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells. MethodsX. 2, 440-445 (2015).
  2. Il Hwang, S., Han, D. K. Subcellular fractionation for identification of biomarkers: Serial detergent extraction by subcellular accessibility and solubility. Methods in Molecular Biology. 1002, 25-35 (2013).
  3. Clayton, D. A., Shadel, G. S. Isolation of mitochondria from cells and tissues. Cold Spring Harbor Protocols. 2014 (10), 1040-1041 (2014).
  4. Stimpson, S. E., Coorssen, J. R., Myers, S. J. Optimal isolation of mitochondria for proteomic analyses. Analytical Biochemistry. 475, 1-3 (2015).
  5. Sundström, C., Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). International Journal of Cancer. 17 (5), 565-577 (1976).
  6. Hodge, T., Colombini, M. Regulation of metabolite flux through voltage-gating of VDAC channels. Journal of Membrane Biology. 157 (3), 271-279 (1997).
  7. Therien, A. G., Blostein, R. Mechanisms of sodium pump regulation. American journal of physiology. Cell physiology. 279 (3), 541-566 (2000).
  8. Towbin, H., Staehelin, T., Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of the America. 76 (9), 4350-4354 (1979).
  9. Barber, R. D., Harmer, D. W., Coleman, R. A., Clark, B. J. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiological Genomics. 21 (3), 389-395 (2005).
  10. Bradbury, E. M., Cary, P. D., Crane-Robinson, C., Rattle, H. W. E. Conformations and interactions of histones and their role in chromosome structure. Annals of the New York Academy of Sciences. 222, 266-289 (1973).
  11. McCaig, W. D., Deragon, M. A., Haluska, R. J., Hodges, A. L., Patel, P. S., LaRocca, T. J. Cell fractionation of U937 cells in the absence of high-speed centrifugation. Journal of Visualized Experiments. (143), e59022 (2019).
  12. McCaig, W. D., et al. Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis. Cell Death Discovery. 4, 55 (2018).

Erratum

Erratum: Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

An erratum was issued for: Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification. The Authors section was updated.

The Authors section was updated from:

William McCaig1
Timothy LaRocca1
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

to:

William D. McCaig1
Matthew A. Deragon1
Phillip V. Truong1
Angeleigh R. Knapp1
Keven J. Hughes1
Timothy J. LaRocca1
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

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