Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

Summary

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.

Abstract

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.

Introduction

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-based^1,2, relying on the use of buffers containing increas....

Protocol

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 (100x).
   3. Add 91.9 mg of sodium orthovanadate (SOV) to 1 mL of deionized water to prepare a 500 mM stock.
      NOTE: Wear protective equipm....

Results

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

Discussion

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

Materials

List of materials used in this article

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