Name: nitrogen cavitation and differential centrifugation allows for monitoring the distribution of peripheral membrane proteins in cultured cells
Uploaded: 2017-08-18T14:00:00
Description: Watch this Scientific Journal Video about Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells at JoVE.com

This work was funded by GM055279, CA116034 and CA163489.

1. Buffer and Equipment Preparations
<ol>
	<li>Chill 45 mL cell disruption bombs, 15 mL tubes, and ultracentrifugation tubes at 4 &#176;C.</li>
	<li>Prepare and chill 25 mL of homogenization buffer per 2 x 107 cells at 4 &#176;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 MgCl<su...

<ol>
	<li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+on+the+move:+insights+gained+from+fluorescent+protein+technologies.">Proteins on the move: insights gained from fluorescent protein technologies.</a> Nat Rev Mol Cell Biol. 12 (10), 656-668 (2011).</li><li>Bunger, S., Roblick, U. J., Habermann, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+commercial+extraction+kits+for+subsequent+membrane+protein+profiling.">Comparison of five commercial extraction kits for subsequent membrane protein profiling.</a> Cytotechnology. 61 (3), 153-159 (2009).</li><li>Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+cultured+cells+by+nitrogen+cavitation.">Disruption of cultured cells by nitrogen cavitation.</a> Cold Spring Harb Protoc. (11), (2010).</li><li>Klempner, M. S., Mikkelsen, R. B., Corfman, D. H., Andre-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+plasma+membranes.+I.+High-yield+purification+of+human+neutrophil+plasma+membrane+vesicles+by+nitrogen+cavitation+and+differential+centrifugation.">Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.</a> J Cell Biol. 86 (1), 21-28 (1980).</li><li>Philips, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+molecular+weight+GTP-binding+proteins+in+human+neutrophil+granule+membranes.">Low molecular weight GTP-binding proteins in human neutrophil granule membranes.</a> Journal of Biological Chemistry. 266, 1289-1298 (1991).</li><li>Wallach, D. F., Soderberg, J., Bricker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phospholipides+of+Ehrlich+ascites+carcinoma+cells:+composition+and+intracellular+distribution.">The phospholipides of Ehrlich ascites carcinoma cells: composition and intracellular distribution.</a> Cancer Res. 20, 397-402 (1960).</li><li>Hunter, M. J., Commerford, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+homogenization+of+mammalian+tissues.">Pressure homogenization of mammalian tissues.</a> Biochim Biophys Acta. 47, 580-586 (1961).</li><li>Wallach, D. F., Kamat, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+Cytoplasmic+Membrane+Fragments+from+Ehrlich+Ascites+Carcinoma.">Plasma and Cytoplasmic Membrane Fragments from Ehrlich Ascites Carcinoma.</a> Proc Natl Acad Sci U S A. 52, 721-728 (1964).</li><li>Birckbichler, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preperation+of+plasma+membrane+vesicles+by+nitrogen+cavitation.">Preperation of plasma membrane vesicles by nitrogen cavitation.</a> TCA manual / Tissue Culture Association. 3 (3), 653-654 (1977).</li><li>Brock, T. G., Paine, R., Peters-Golden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+5-lipoxygenase+to+the+nucleus+of+unstimulated+rat+basophilic+leukemia+cells.">Localization of 5-lipoxygenase to the nucleus of unstimulated rat basophilic leukemia cells.</a> J Biol Chem. 269 (35), 22059-22066 (1994).</li><li>Dowben, R. M., Gaffey, A., Lynch, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+liver+and+muscle+polyribosomes+in+high+yield+after+cell+disruption+by+nitrogen+cavitation.">Isolation of liver and muscle polyribosomes in high yield after cell disruption by nitrogen cavitation.</a> FEBS Letters. 2 (1), (1968).</li><li>Dai, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+prenylcysteine+carboxyl+methyltransferase+is+in+the+endoplasmic+reticulum.">Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum.</a> J Biol Chem. 273 (24), 15030-15034 (1998).</li><li>Wynne, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rap1-interacting+adapter+molecule+(RIAM)+associates+with+the+plasma+membrane+via+a+proximity+detector.">Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector.</a> J Cell Biol. , (2012).</li><li>Zhou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VPS35+binds+farnesylated+N-Ras+in+the+cytosol+to+regulate+N-Ras+trafficking.">VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking.</a> J Cell Biol. 214 (4), 445-458 (2016).</li><li>Sim, D. S., Dilks, J. R., Flaumenhaft, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+possess+and+require+an+active+protein+palmitoylation+pathway+for+agonist-mediated+activation+and+in+vivo+thrombus+formation.">Platelets possess and require an active protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus formation.</a> Arterioscler Thromb Vasc Biol. 27 (6), 1478-1485 (2007).</li><li>Pace, P. E., Peskin, A. V., Han, M. H., Hampton, M. B., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxidized+peroxiredoxin+2+interacts+with+the+protein+disulfide-+isomerase+ERp46.">Hyperoxidized peroxiredoxin 2 interacts with the protein disulfide- isomerase ERp46.</a> Biochem J. 453 (3), 475-485 (2013).</li><li>Annis, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+localized+Bcl-2+prevents+apoptosis+when+redistribution+of+cytochrome+c+is+a+late+event.">Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.</a> Oncogene. 20 (16), 1939-1952 (2001).</li><li>Berkowitz, S. A., Wolff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+calcium+sensitivity+of+tubulin+polymerization.+The+contributions+of+temperature,+tubulin+concentration,+and+associated+proteins.">Intrinsic calcium sensitivity of tubulin polymerization. The contributions of temperature, tubulin concentration, and associated proteins.</a> J Biol Chem. 256 (21), 11216-11223 (1981).</li></ol>

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 ...

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 &#945;-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 they are catabolized. Peripheral membrane proteins are secondarily driven to membranes, usually as a consequence of post-translational modification with hydrophobic molecules such as lipids. In contrast to integral membrane proteins, the association of peripheral membrane proteins with cellular membranes is reversible and can be regulated. Many peripheral membrane proteins function in signaling pathways, and regulated association with membranes is one mechanism for activating or inhibiting a pathway. One example of a signaling molecule that is a peripheral membrane protein is the small GTPase, RAS. After a series of post-translational modifications that include modification with a farnesyl lipid, the modified C-terminus of a mature RAS protein inserts into the cytoplasmic leaflet of the cellular membrane. Specifically, the plasma membrane is where the RAS engages its downstream effector RAF1. To prevent constitutive activation of mitogen-activated protein kinase (MAPK) pathway, multiple levels of control of RAS are in place. Besides rendering RAS inactive by hydrolyzing GTP into GDP, active RAS also can be released from the plasma membrane by modifications or interactions with solubilizing factors to inhibit signaling. Although fluorescent live imaging affords cell biologists the opportunity to observe the subcellular localization of fluorescent protein-tagged peripheral membrane proteins1, there remains a critical need to evaluate membrane association of endogenous proteins semi-quantitatively with simple biochemical approaches.
The proper biochemical evaluation of protein partitioning between membrane and soluble fractions is critically dependent on two factors: cellular homogenization and efficient separation of membrane and soluble fractions. Although some protocols, including the most widely used commercialized kits, depend on detergent-based cell homogenization, these methods can obfuscate analysis by extracting membrane proteins into the soluble phase2. Accordingly, non-detergent based, mechanical methods of cell disruption provide cleaner results. There are several methods of mechanical disruption of cells grown in culture or harvested from blood or organs. These include Dounce homogenization, fine needle disruption, ball-bearing homogenization, sonication and nitrogen cavitation. Here we evaluate nitrogen cavitation and compare it to other methods. Nitrogen cavitation relies on nitrogen that is dissolved in the cytoplasm of the cells under high pressure. After equilibration, the cell suspension is abruptly exposed to atmospheric pressure such that nitrogen bubbles are formed in the cytoplasm that tear open the cell as a consequence of their effervescence. If the pressure is sufficiently high, nitrogen effervescence can disrupt the nucleus3 and membrane bound organelles like lysosomes4. However, if the pressure is kept low enough, the decompression will disrupt the plasma membrane and ER but not other organelles, thereby spilling both cytosol and intact cytoplasmic organelles into the homogenate that is designated the cavitate5. For this reason, nitrogen cavitation is the method of choice for isolating organelles like lysosomes and mitochondria.
However, it is also an excellent way of preparing a homogenate that can be easily separated into membrane and soluble fractions. The pressure vessel (henceforth called &#34;the bomb&#34;) used during cavitation consists of a thick stainless steel casing that withstands high pressure, with an inlet for delivery of the nitrogen gas from a tank and an outlet port with an adjustable discharge valve.
Nitrogen cavitation has been used for cell homogenization since the 1960s6. In 1961, Hunter and Commerfold7 established nitrogen cavitation as a viable option for mammalian tissue disruption. Since then, researchers have adapted the technique to various cells and tissues with success, and nitrogen cavitation has become a staple in multiple applications, including membrane preparation8,9, nuclei and organelle preparation10,11, and labile biochemical extraction. Currently, cell biologists more often employ other methods of cell homogenization because the benefits of nitrogen homogenization have not been widely advertised, nitrogen bombs are expensive and there is a misconception that a relatively large number of cells is required. Protocols for nitrogen cavitation to achieve cell-free homogenates with intact nuclei have not been published, and in most published evaluations volumes of 20 mL of cell suspension were used. To adapt this classic technique to suit current requirements of working with small-scale samples, we present a modified protocol of nitrogen cavitation specifically designed for cultured cells. After nitrogen cavitation, the homogenate is separated into soluble (S) and membrane (P) fractions by differential centrifugation, first with a low-speed spin to remove nuclei and unbroken cells, and then with a high-speed spin (&#62;100,000 x g) to separate membranes from the soluble fraction. We analyze the efficiency of the separation with immunoblots and compare nitrogen cavitation with other mechanical disruption techniques. We also investigate the osmotic effect of homogenization buffer during nitrogen cavitation.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Cell Disruption Vessel (45 mL)</td>
			<td style="width:181px;">Parr Instrument</td>
			<td style="width:145px;">4639</td>
			<td style="width:296px;">Nitrogen cavitation Bomb</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Dounce homogenizer (2 mL)</td>
			<td style="width:181px;">Kontes</td>
			<td style="width:145px;">885300-0002</td>
			<td style="width:296px;">Dounce pestle and tube</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">U-100 Insulin Syringe 28G&#189;</td>
			<td style="width:181px;">Becton Dickinson</td>
			<td style="width:145px;">329461</td>
			<td style="width:296px;">Needle</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Atg12 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">271688</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">&#946;-actin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">47778</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">&#946;-tubulin antibody</td>
			<td style="width:181px;">DSHB</td>
			<td style="width:145px;">E7-s</td>
			<td style="width:296px;">Mouse antibody, use at 1:5000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Calnexin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">23954</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Calregulin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">373863</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Catalase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">271803</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">CIMPR antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:145px;">124767</td>
			<td style="width:296px;">Rabbit antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">EEA1 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">137130</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">EGFR antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">373746</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">F0-ATPase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">514419</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">F1-ATPase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">55597</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Fibrillarin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">374022</td>
			<td style="width:296px;">Mouse antibody, use at 1:200 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Golgin 97 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">59820</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">HDAC1 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">81598</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Hexokinase 1 antibody</td>
			<td style="width:181px;">Cell Signaling Technology</td>
			<td style="width:145px;">2024S</td>
			<td style="width:296px;">Rabbit antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Lamin A/C antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">376248</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">LAMP1 antibody</td>
			<td style="width:181px;">DSHB</td>
			<td style="width:145px;">H4A3-c</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Na+/K+ ATPase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">48345</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Rab7 antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:145px;">137029</td>
			<td style="width:296px;">Rabbit antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Rab9 antibody</td>
			<td style="width:181px;">Thermo</td>
			<td style="width:145px;">MA3-067</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">RCAS1 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">398052</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">RhoGDI antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">360</td>
			<td style="width:296px;">Rabbit antibody, use at 1:3000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Ribosomal protein S6 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">74459</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Sec61a antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">12322</td>
			<td style="width:296px;">Goat antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Thickwall Polycarbonate ultracentrifuge tube</td>
			<td style="width:181px;">Beckman Coulter</td>
			<td style="width:145px;">349622</td>
			<td style="width:296px;">Sample tube for ultracentrifugation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">TLK-100.3 rotor</td>
			<td style="width:181px;">Beckman Coulter</td>
			<td style="width:145px;">349481</td>
			<td style="width:296px;">rotor for ultracentrifugation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Optima MAX High-Capacity Personal Ultracentrifuge</td>
			<td style="width:181px;">Beckman Coulter</td>
			<td style="width:145px;">364300</td>
			<td style="width:296px;">ultracentrifuge</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">cOmplete protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>11697498001</td>
			<td>protease inhibitors</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Cell Scrapers with 25cm Handle and 3.0cm Blade</td>
			<td>Corning</td>
			<td>353089</td>
			<td>large cell scraper</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Magnetic Stir Bar</td>
			<td>Fisher Scientific</td>
			<td>14-513-57SIX</td>
			<td>micro stir bar</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Ceramic-Top Magnetic Stirrer</td>
			<td>Fisher Scientific</td>
			<td>S504501AS</td>
			<td>magnetic stirrer</td>
		</tr>
	</tbody>
</table>

nitrogen cavitation and differential centrifugation allows for monitoring the distribution of peripheral membrane proteins in cultured cells

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.

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...

Watch this Scientific Journal Video about Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells at JoVE.com

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

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.

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

The overall goal of this experimental procedure, is to optimally homogenize cultured cells and to analyze partitioning of cellular proteins between membrane and soluble fractions. This method can help address one of the key challenges in cell biology, that is the subcellular compartmentalization of membrane peripheral proteins. The main advantage of this technique is, it can reproducibly detect partitioning of endogenous proteins, inside a solid and membrane fractions, with great accuracy and without use of detergents.To begin, culture HGK293 cells at 90%confluency, such that the yield per one 15 centimeter dish is about 2 x 10 to the 7th cells when cultured in DMEM at 37 degrees celsius, with 5%carbon dioxide. Then, aspirate the growth medium by vacuum. Next, place the culture dishes on ice and gently wash the cells twice on the culture dishes with 10 milliliters of chilled homogenization buffer per dish.After washing, use a large cell scrapper to harvest the cells, using an appropriate volume of homogenization buffer. Now, transfer the cell suspension to a clean, pre-chilled self destruction bomb, placed in an ice bath on a stir plate. To maintain suspension homogeneity, place a micro magnetic stir bar inside the bomb and turn on the stir plate.After that, add one protease inhibitor tablet to the suspension and close the bomb according to the manufacturer's instructions, including a crucial step of pushing the O ring upward to secure proper closing of the bomb. Once done, pressurize the bomb gradually with the nitrogen gas tank, as per the manufacturer's instructions, until the pressure gauge reads between 300 to 600 PSI. After the desired pressure is obtained, close all valves and disconnect the nitrogen tank.Allow 20 minutes for the nitrogen to dissolve and reach equilibrium within the cells. Next, use a disposable wipe to remove excess water around the discharge valve, and gently open the valve to achieve a drop wise release of homogenate, collect the homogenate in a pre-chilled 15 milliliter tube. Observe the milky appearance with foam on top of the cavitate.During the pressurization, it is critical to avoid simple loss by collecting the cavitate with slow, valvular discharge into a collection tube with an adequate access volume capacity that is optimally positioned to accommodate sudden discharge near the end of collection. After collecting the cavitate, centrifuge the tubes to remove unbroken cells in nuclei. And then collect the post-nuclear supernatant or PNS.Next, transfer the PNS to a polycarbonate ultracentrifuge tube. Perform ultracentrifugation to separate the cytosolic and membrane fractions. After ultra centrifugation and separation of pellet and supernatant.Use a one milliliter pipette to collect the supernatant or S fraction. After collecting the supernatant, rinse the pellet carefully with four milliliters of cold PBS, taking care not to disturb pellet and then remove the buffer by aspiration. Re-suspend the pellet, using the same volume of solubilization buffer, as the cytosolic fraction to achieve cell equivalence.Next, centrifuge the fully solubilized pellet suspension in a tabletop centrifuge. Use a one milliliter pipette to collect the supernatant or P fraction and discard the pellet. Finally, store the fractions at 80 degree celsius.Endogenous protein levels from both cytosolic and membrane fractions, isolated from three representative cell lines, are shown after western blot analysis. The presence of loosely associated peripheral membrane proteins such as, EEA1, Rab7, Rab9 and hexokinase 1 in both cytosolic and membrane fractions, demonstrate the utility of this technique and evaluating the strength of the membrane association of peripheral proteins. Via mounts of endogenous proteins in the PNS, were analyzed after subjecting jurkat cells to different mechanical disruption techniques.The yield of peripheral membrane proteins such as, hexokinase 1 and Ras, is higher in samples prepared with nitrogen cavitation. The homogenisation efficiency was compared between a hypotonic buffer and a buffer made isotonic with sucrose or sodium chloride. The protein levels in the PNS of cells disrupted using isotonic sucrose buffer, showed less recovery except for fibrollarin, which was enriched.These results indicate the partitioning of farnesylated NRAS into both P and S fractions. The absence of the preno modification of NRAS results in entirely cytosolic localisation, which therefore, confirms the reliability of this protocol for studying the dynamics of peripheral membrane protein partitioning. Once mastered, the separation of cytosolic and membrane fractions without re-suspension of the membrane fraction can be performed in an hour and 45 minutes or about 4 hours with re-suspension of the membrane fraction.Due to the inherent artifactual nature of bichemical fractionation, it is important to focus on the changes across experimental conditions in the partitioning of proteins, rather than the absolute distribution between cytosolic and membrane fractions. Additionally, we suggest testing different homogenization buffers for nitrogen cavitation to achieve optimal results. Post this procedure, immunoblotting and enzyme activity assays can be performed in order to answer additional questions like, membrane associated strength of peripheral proteins.Don't forget that working with high pressure nitrogen bombs can be extremely hazardous and precautions such as, wearing appropriate personal protection should always be taken while performing this procedure. Thanks for watching, good luck with your experiments.

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