Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

Summary

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

Abstract

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Introduction

Reliable identification of protein localization is often necessary when examining molecular pathways in eukaryotic cells. Methods to obtain subcellular fractions are utilized by researchers to more closely examine cellular components of interest.

The majority of existing cell fractionation methods generally fall into two broad categories, detergent-based^1,2 and ultracentrifugation-based^3,4,5, which can be differentiated by speed, precision and cost. Detergent based protocols rely on the use....

Protocol

1. Prepare Buffers and Reagents

NOTE: See Table 1.

1. Prepare solutions of buffer A, lysis buffer B, sample buffer and digitonin.
   1. Prepare buffer A by adding 8.77 g of NaCl and 50 mL of HEPES (1 M, pH 7.4) to 900 mL deionized water, adjust final volume to 1 L with deionized water.
      NOTE: Final concentrations are 150 mM NaCl and 50 mM HEPES.
   2. Prepare lysis buffer B by adding 20 mL of HEPES (1 M, pH 7.4), 0.75 g of KCl, 0.19 g of MgCl₂, 2 mL of Ethylenediaminetetraacetic acid (0.5 M EDTA), 2 mL of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0....

Results

Successful fractionation of undifferentiated U937⁸ cells grown in suspension was accomplished using the protocol detailed above and illustrated in Figure 1. The samples obtained with this method were subjected to western blotting⁹ utilizing a wet transfer method to a polyvinylidene fluoride (PVDF) membrane. The membrane was subsequently probed with antibodies against cytoplasmic, mitochondrial and membrane local.......

Discussion

The development of this protocol arose from an inability to separate mitochondrial and membrane samples, using commercially available kits, for analysis of protein localization during necroptosis¹⁴. The primary limitations of premade kits are their inability to be adapted to the needs of individual researchers, their cost per sample and limited number of samples able to be processed. The method presented here can be performed without the use of expensive reagents and without the necessity for expe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Digitonin	TCI Chemicals	D0540	For Cytoplasm Extraction
D-Mannitol	Sigma-Aldrich	M4125	For Lysis buffer B
Dounce homogenizer	VWR	22877-282	For Homogenization
end-over-end rotator	Barnstead	N/A	For Cytoplasm Extraction
ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)	Alfa Aesar	J61721	For Lysis buffer B
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E7889	For Lysis buffer B
GAPDH (14C10)	Cell Signalling Technologies	2118	For detection of cytoplasmic fractions on western blot, dilution: 1:10000
HEPES	VWR	J848	For Lysis buffers A and B
KCl	Sigma-Aldrich	P9541	For Lysis buffer B
MgCl2	Alfa Aesar	12315	For Lysis buffer B
Na, K-ATPase a1 (D4Y7E)	Cell Signalling Technologies	23565	For detection of plasma membrane fractions on western blot, dilution: 1:1000
NaCl	Sigma-Aldrich	793566	For Lysis buffer A
phenylmethanesulfonyl fluoride (PMSF)	VWR	M145	For Cytoplasm Extraction and Homogenization Buffer
probe sonicator	Qsonica	Q125-110	For Final Samples
Protease Inhibitor Cocktail, General Use	VWR	M221-1ML	For Cytoplasm Extraction
refrigerated centrifuge	Beckman-Coulter	N/A
Sodium dodecyl sulfate (SDS)	VWR	227	For Sample buffer
sodium orthovanadate (SOV)	Sigma-Aldrich	450243	For Lysis buffers A and B
Sucrose	Sigma-Aldrich	S0389	For Lysis buffer B
Tris-buffered Saline (TBS)	VWR	788	For Sample buffer
VDAC (D73D12)	Cell Signalling Technologies	4661	For detection of mitochondrial fractions on western blot, dilution: 1:1000