Method Article

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

DOI:

10.3791/56037

⸱

August 18th, 2017

 , 
1Langone Medical Center, New York University

In This Article

Summary

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Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

Abstract

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Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Introduction

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Cellular proteins can be divided into two classes: those that are associated with membranes and those that are not. Non-membrane associated proteins are found in the cytosol, nucleoplasm and lumina of organelles such as the endoplasmic reticulum (ER). There are two classes of membrane-associated proteins, integral and peripheral. Integral membrane proteins are also known as transmembrane proteins because one or more segments of the polypeptide chain spans the membrane, typically as an α-helix composed of hydrophobic amino acids. Transmembrane proteins are co-translationally inserted into membranes in the course of their biosynthesis and remain so configured until....

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Protocol

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1. Buffer and Equipment Preparations

  1. Chill 45 mL cell disruption bombs, 15 mL tubes, and ultracentrifugation tubes at 4 °C.
  2. Prepare and chill 25 mL of homogenization buffer per 2 x 107 cells at 4 °C. Add one protease inhibitor tablet just before use.
    NOTE: Homogenization buffers typically contain KCl rather than NaCl to better reflect intracellular salt composition. Homogenization buffer used in this protocol consists of 10 mM HEPES at pH 7.4, 10 mM KCl and 1.5 mM MgCl2 (hereinafter referred to as hypotonic homogenization buffer). Most buffers can be adapted for nitrogen cavitation (see Discussion

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Results

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Figure 2 shows the partitioning of cellular proteins from PNS into either the soluble cytosolic fraction (S) or membrane pellet fraction (P). We examined three representative cell lines from different cell types: HEK-293 (epithelial), NIH-3T3 (fibroblast), and Jurkat (lymphocyte). Rho Guanine Dissociation Inhibitor (RhoGDI) and cation-independent mannose-6-phosphate receptor (CIMPR) were used as positive controls for cytosolic and membrane fractions, respecti.......

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Discussion

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The advantages of nitrogen cavitation over other methods of mechanical disruption are manifold. Perhaps the most significant benefit is its ability to gently yet efficiently homogenize specimens. The physical principles of decompression cools samples instead of generating local heating damage like ultrasonic and friction/shearing based techniques. Cavitation is also extremely efficient at disrupting the plasma membrane. Because nitrogen bubbles are generated within each individual cell upon decompression, the cavitation .......

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Disclosures

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The authors declare that they have no competing financial interests.

Acknowledgements

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This work was funded by GM055279, CA116034 and CA163489.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Cell Disruption Vessel (45 mL)Parr Instrument4639Nitrogen cavitation Bomb
Dounce homogenizer (2 mL)Kontes885300-0002Dounce pestle and tube
U-100 Insulin Syringe 28G½Becton Dickinson329461Needle
Atg12 antibodySanta Cruz271688Mouse antibody, use at 1:1000 dilution
β-actin antibodySanta Cruz47778Mouse antibody, use at 1:1000 dilution
β-tubulin antibodyDSHBE7-sMouse antibody, use at 1:5000 dilution
Calnexin antibodySanta Cruz23954Mouse antibody, use at 1:1000 dilution
Calregulin antibodySanta Cruz373863Mouse antibody, use at 1:1000 dilution
Catalase antibodySanta Cruz271803Mouse antibody, use at 1:1000 dilution
CIMPR antibodyAbcam124767Rabbit antibody, use at 1:1000 dilution
EEA1 antibodySanta Cruz137130Mouse antibody, use at 1:1000 dilution
EGFR antibodySanta Cruz373746Mouse antibody, use at 1:1000 dilution
F0-ATPase antibodySanta Cruz514419Mouse antibody, use at 1:1000 dilution
F1-ATPase antibodySanta Cruz55597Mouse antibody, use at 1:1000 dilution
Fibrillarin antibodySanta Cruz374022Mouse antibody, use at 1:200 dilution
Golgin 97 antibodySanta Cruz59820Mouse antibody, use at 1:1000 dilution
HDAC1 antibodySanta Cruz81598Mouse antibody, use at 1:1000 dilution
Hexokinase 1 antibodyCell Signaling Technology2024SRabbit antibody, use at 1:1000 dilution
Lamin A/C antibodySanta Cruz376248Mouse antibody, use at 1:1000 dilution
LAMP1 antibodyDSHBH4A3-cMouse antibody, use at 1:1000 dilution
Na+/K+ ATPase antibodySanta Cruz48345Mouse antibody, use at 1:1000 dilution
Rab7 antibodyAbcam137029Rabbit antibody, use at 1:1000 dilution
Rab9 antibodyThermoMA3-067Mouse antibody, use at 1:1000 dilution
RCAS1 antibodySanta Cruz398052Mouse antibody, use at 1:1000 dilution
RhoGDI antibodySanta Cruz360Rabbit antibody, use at 1:3000 dilution
Ribosomal protein S6 antibodySanta Cruz74459Mouse antibody, use at 1:1000 dilution
Sec61a antibodySanta Cruz12322Goat antibody, use at 1:1000 dilution
Thickwall Polycarbonate ultracentrifuge tubeBeckman Coulter349622Sample tube for ultracentrifugation
TLK-100.3 rotorBeckman Coulter349481rotor for ultracentrifugation
Optima MAX High-Capacity Personal UltracentrifugeBeckman Coulter364300ultracentrifuge
cOmplete protease inhibitor cocktail tabletsRoche11697498001protease inhibitors
Cell Scrapers with 25cm Handle and 3.0cm BladeCorning353089large cell scraper
Magnetic Stir BarFisher Scientific14-513-57SIXmicro stir bar
Ceramic-Top Magnetic StirrerFisher ScientificS504501ASmagnetic stirrer

References

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  1. Miyawaki, A. Proteins on the move: insights gained from fluorescent protein technologies. Nat Rev Mol Cell Biol. 12 (10), 656-668 (2011).
  2. Bunger, S., Roblick, U. J., Habermann, J. K. Comparison of five commercial extraction kits for subsequen....

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Tags

Nitrogen CavitationDifferential CentrifugationPeripheral Membrane ProteinsSubcellular FractionationUltracentrifugationCytosolic FractionMembrane FractionProtein PartitioningCell HomogenizationWestern Blot Analysis

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