Work was supported by NIH-1R15HL135675-01 to Timothy J. LaRocca

1. Prepare Buffers and Reagents
NOTE: See Table 1.
<ol>
	<li>Prepare solutions of buffer A, lysis buffer B, sample buffer and digitonin.
	<ol>
		<li>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.</li>
		<li>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(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (0.5 M EGTA), 38.26 g of mannitol and 23.96 g of sucrose to 900 mL of deionized water, adjust final volume to 1 L with deionized water. 
		NOTE: Final concentrations are 20 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 210 mM mannitol and 70 mM sucrose.</li>
		<li>Prepare sample buffer by adding 0.01 g of sodium dodecyl sulfate (SDS) to 10 mL of tris-buffered saline (TBS) for a final concentration of 0.1% SDS.</li>
		<li>Prepare a stock solution of digitonin by adding 25 mg of digitonin to 100 mL of deionized water (final concentration is 250 &#181;g/mL).</li>
		<li>Store all buffer solutions at 4 &#176;C and digitonin at -20 &#176;C until the start of experiment.</li>
	</ol>
	</li>
	<li>Prepare fresh solutions of protease and phosphatase inhibitors to be added to buffer solutions prior to addition to cells.
	<ol>
		<li>Prepare a stock solution of phenylmethanesulfonyl fluoride (PMSF) by adding 17.4 mg of PMSF to 1 mL of 100% ethanol (final concentration is 100 mM). 
		CAUTION: Wear appropriate protective equipment and exercise caution when handling PMSF. PMSF is hazardous if ingested and slightly hazardous in case of skin contact (irritant), eye contact (irritant) or inhalation; it is corrosive to eyes and skin.</li>
		<li>Prepare a commercially available protease inhibitor cocktail (100&#215;) according to the manufacturer&#8217;s instructions (see the Table of Materials).</li>
		<li>Prepare a stock solution of sodium orthovanadate (SOV) by adding 92 mg of SOV to 1 mL of deionized water (final concentration is 500 mM). 
		CAUTION: Wear appropriate protective equipment and use caution when handling. SOV is hazardous in case of eye contact (irritant), ingestion or inhalation. Severe over-exposure can result in death.</li>
	</ol>
	</li>
</ol>
2. PBS Wash
<ol>
	<li>Concentrate and wash cells in phosphate-buffered saline (PBS) prior to fractionation.
	<ol>
		<li>Centrifuge cell suspension at an appropriate speed to create a pellet. For example, centrifuge a suspension of U937 cells at 400 &#215; g for 10 min.</li>
		<li>Remove the supernatant, resuspend cell pellet in room-temperature PBS at a final concentration of 4 &#215; 106 cells/mL and pipette gently to break up clumps.</li>
		<li>Centrifuge the cell suspension at 400 &#215; g for 10 min to pellet cells.</li>
		<li>Remove the supernatant and resuspend cell pellet in ice cold buffer A at a final concentration of 2 &#215; 107 cells/mL. 
		NOTE: All subsequent steps should be carried out at 4 &#176;C or on ice and all buffers should be pre-chilled.</li>
	</ol>
	</li>
</ol>
3. Cytosolic Protein Isolation
<ol>
	<li>Extract cytosolic proteins by incubation with the detergent digitonin.
	<ol>
		<li>Immediately prior to resuspension of cells (step 3.1.3) add 10 &#181;L of stock PMSF (100 mM), 10 &#181;L of protease Inhibitor (100&#215;), 2 &#181;L of stock SOV (500 mM) and 100 &#181;L of stock digitonin (250 &#181;g/mL) to 878 &#181;L of buffer A (final concentrations are 1 mM PMSF, 1&#215; Protease Inhibitor, 1 mM SOV and 25 &#181;g/mL digitonin; adjust the final volume as per the number of cells being used). Keep the solution on ice until addition to cell pellet.</li>
		<li>Centrifuge the cell suspension at 400 &#215; g for 10 min and remove the supernatant.</li>
		<li>Resuspend the cell pellet in buffer A solution containing inhibitors and digitonin (prepared in step 3.1.1) at a final concentration of 2 &#215; 107 cells/mL, pipette gently to break up clumps.</li>
		<li>Incubate the cell suspension on an end-over-end rotator at 4 &#176;C for 20 min.</li>
		<li>Centrifuge the cell suspension at 400 &#215; g for 10 min. Collect the supernatant and place it in a clean centrifuge tube.</li>
		<li>Centrifuge the collected supernatant at 18,000 &#215; g for 20 min to pellet cellular debris.</li>
		<li>Transfer the supernatant to a clean centrifuge tube.</li>
		<li>Repeat steps 3.1.5 and 3.1.6 until no pellet is obtained following centrifugation.</li>
		<li>Collect the supernatant containing the cytosolic proteins and store it at 4 &#176;C (short term) or -20 &#176;C (long term).</li>
	</ol>
	</li>
	<li>Remove excess digitonin and cytosolic proteins by centrifugation.
	<ol>
		<li>Resuspend the digitonin-permeabilized cell pellet (from step 3.1.5) in buffer A at a final concentration of 4 &#215; 106 cells/mL and pipette gently to break up clumps.</li>
		<li>Centrifuge the digitonin-permeabilized cell suspension at 400 &#215; g for 10 min and remove the supernatant. 
		NOTE: Repeated washes in buffer A can be performed to remove excess cytosolic contaminants.</li>
	</ol>
	</li>
</ol>
4. Cell Homogenization
<ol>
	<li>Incubate the cells on ice in lysis buffer B and lyse them by mechanical means.
	<ol>
		<li>Immediately prior to resuspension of cells (step 4.1.2) add 10 &#181;L of stock PMSF (100 mM) and 2 &#181;L of stock SOV (500 mM) to 988 &#181;L of lysis buffer B (final concentrations are 1 mM PMSF and 1 mM SOV; adjust final volume to accommodate number of cells being lysed) and keep solution on ice until addition to cell pellet.</li>
		<li>Resuspend the cell pellet (from step 3.2.2) in ice cold lysis buffer B solution containing PMSF and SOV (prepared in step 4.1.1) at a final concentration of 4 &#215; 106 cells/mL.</li>
		<li>Incubate the cell suspension on ice for 30 min.</li>
		<li>Transfer the cell suspension to a pre-chilled Dounce homogenizer (with a tight-fitting B pestle) on ice and perform 40 passes with the homogenizer pestle using slow, even strokes. 
		NOTE: Alternatively utilize other means of mechanical cell lysis as detailed in discussion section.</li>
		<li>Collect the homogenate and transfer it to a clean centrifuge tube.</li>
		<li>Wash the homogenizer pestle and tube with a small volume (1 to 2 mL) of lysis buffer B and add it to the homogenate.</li>
		<li>Centrifuge the homogenate at 400 &#215; g (or the minimum speed required to pellet unbroken cells) for 10 min.</li>
		<li>Transfer the supernatant to a clean centrifuge tube. 
		Note: If a significant pellet remains repeat steps 4.1.4 through 4.1.6 to increase the yield of fractions as detailed in the discussion section. The protocol can be paused here, and the homogenate stored at 4 &#176;C for short term (24 h).</li>
	</ol>
	</li>
</ol>
5. Differential Centrifugation
<ol>
	<li>Centrifuge the homogenate at increasing speeds to remove cellular debris, isolate mitochondria and membrane fractions.
	<ol>
		<li>Centrifuge the supernatant (from step 4.1.8) at 500 &#215; g for 10 min. Transfer supernatant to a clean centrifuge tube, and discard any pellet.</li>
		<li>Centrifuge supernatant (from step 5.1.1) at 1,000 &#215; g for 10 min. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.</li>
		<li>Centrifuge the supernatant (from step 5.1.2) at 2,000 &#215; g for 10 min. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.</li>
		<li>Centrifuge the supernatant (from step 5.1.3) at 4,000 &#215; g for 15 min. Transfer supernatant to a clean centrifuge tube, keep pellet containing mitochondria.</li>
		<li>Resuspend the mitochondria pellet in a small volume (0.5&#8210;1 mL) of lysis buffer B.</li>
		<li>Centrifuge the suspended pellet at 4,000 &#215; g for 15 min. Remove the supernatant and resuspend the mitochondrial pellet in the desired final volume of sample buffer (e.g., 250 to 500 &#181;L, depending on the size of the pellet and desired concentration).</li>
		<li>Centrifuge the supernatant (from step 5.1.4) at 4,000 &#215; g for 15 min. Transfer the supernatant to a clean centrifuge tube. Repeat this step until no pellet is obtained following centrifugation.</li>
		<li>Spin the supernatant at 18,000 &#215; g for 3 h.</li>
		<li>Remove the supernatant, and keep the pellet containing membrane proteins. Resuspend the membrane pellet in a small volume (0.5&#8211;1 mL) of lysis buffer B.</li>
		<li>Centrifuge the suspended pellet at 18,000 &#215; g for 1 h.</li>
		<li>Remove the supernatant and resuspend the membrane pellet in the desired final volume of sample buffer (250 to 500 &#181;L, depending on the size of the pellet and desired concentration).</li>
	</ol>
	</li>
	<li>Sonicate the sample pellets for 3 s in an ice bath at a power setting of 5 (50% of 125 W maximum power at 20 kHz, see the Table of Materials).</li>
	<li>Store the samples at 4 &#176;C (short term) or -20 &#176;C (long term).</li>
	<li>Examine the samples for purity of fractionation by performing a western blot utilizing antibodies against protein markers found in the cytoplasm, mitochondria and membrane compartments of the cell (refer to the representative results section).</li>
</ol>

<ol>
	<li>Baghirova, S., Hughes, B. G., Hendzel, M. J., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+fractionation+and+isolation+of+subcellular+proteins+from+tissue+or+cultured+cells.">Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.</a> MethodsX. 2, e440-e445 (2015).</li><li>Hwang, S., Han, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+for+identification+of+biomarkers:+Serial+detergent+extraction+by+subcellular+accessibility+and+solubility.">Subcellular fractionation for identification of biomarkers: Serial detergent extraction by subcellular accessibility and solubility.</a> Methods in Molecular Biology. 1002, 25-35 (2013).</li><li>Song, Y., Hao, Y., 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=Sample+preparation+project+for+the+subcellular+proteome+of+mouse+liver.">Sample preparation project for the subcellular proteome of mouse liver.</a> Proteomics. 6 (19), 5269-5277 (2006).</li><li>Lenstra, J. A., Bloemendal, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+the+total+protein+population+from+cultured+cells+upon+fractionation+by+chemical+extractions.">Topography of the total protein population from cultured cells upon fractionation by chemical extractions.</a> European Journal of Biochemistry. 135 (3), 413-423 (1983).</li><li>Michelsen, U., von Hagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+19+Isolation+of+Subcellular+Organelles+and+Structures.">Chapter 19 Isolation of Subcellular Organelles and Structures.</a> Methods in Enzymology. 463 (C), 305-328 (2009).</li><li>Stimpson, S. E., Coorssen, J. R., Myers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+isolation+of+mitochondria+for+proteomic+analyses).">Optimal isolation of mitochondria for proteomic analyses).</a> Analytical Biochemistry. 475, 1-3 (2015).</li><li>Williamson, C. D., Wong, D. S., Bozidis, P., Zhang, A., Colberg-Poley, A. 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+Endoplasmic+Reticulum,+Mitochondria,+and+Mitochondria-Associated+Membrane+and+Detergent+Resistant+Membrane+Fractions+from+Transfected+Cells+and+from+Human+Cytomegalovirus-Infected+Primary+Fibroblasts.">Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.</a> Current protocols in cell biology. 68, (2015).</li><li>Sundstr&#246;m, C., Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+histiocytic+lymphoma+cell+line+(U-937).">Establishment and characterization of a human histiocytic lymphoma cell line (U-937).</a> International Journal of Cancer. 17 (5), 565-577 (1976).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences. 76 (9), 4350-4354 (1979).</li><li>Barber, R. D., Harmer, D. W., Coleman, R. A., Clark, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAPDH+as+a+housekeeping+gene:+analysis+of+GAPDH+mRNA+expression+in+a+panel+of+72+human+tissues.">GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.</a> Physiological Genomics. 21 (3), 389-395 (2005).</li><li>Hodge, T., Colombini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+metabolite+flux+through+voltage-gating+of+VDAC+channels.">Regulation of metabolite flux through voltage-gating of VDAC channels.</a> Journal of Membrane Biology. 157 (3), 271-279 (1997).</li><li>Therien, a. G., Blostein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sodium+pump+regulation.+American+journal+of+physiology.">Mechanisms of sodium pump regulation. American journal of physiology.</a> Cell physiology. 279 (3), C541-C566 (2000).</li><li>Devarajan, P., Stabach, P. R., De Matteis, M. A., Morrow, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na,K-ATPase+transport+from+endoplasmic+reticulum+to+Golgi+requires+the+Golgi+spectrin-ankyrin+G119+skeleton+in+Madin+Darby+canine+kidney+cells.">Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (20), 10711-10716 (1997).</li><li>McCaig, W. D., Patel, P. S., 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=Hyperglycemia+potentiates+a+shift+from+apoptosis+to+RIP1-dependent+necroptosis.">Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.</a> Cell Death Discovery. 4, 55 (2018).</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=Homogenization+of+Mammalian+Tissue.">Homogenization of Mammalian Tissue.</a> Cold Spring Harbor Protocols. 2010 (7), (2010).</li></ol>

The authors declare no conflict of interest

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...

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-based1,2 and ultracentrifugation-based3,4,5, which can be differentiated by speed, precision and cost. Detergent based protocols rely on the use of buffers with increasing detergent strength to solubilize distinct components of the cell. This is a rapid and convenient method for processing samples and can be cost effective if the number and size of samples are small. Detergent-based kits can be purchased to isolate cytoplasmic, membrane/organelle (mixed fraction), and nuclear fractions from cells. However, several drawbacks associated with these kits limit their usefulness to researchers. They are designed to easily isolate one or two components of the cell, but are incapable of isolating all fractions from a sample concurrently. The use of detergents means that the plasma membrane and membrane-enclosed organelles will be equally solubilized and, therefore, unable to be separated from one another. An additional complication arises from the proprietary components in these kits which prevents researchers from altering conditions for specific applications. Lastly, they are limited in number of uses and may be prohibitively expensive for larger scale experiments. Non-detergent based kits exist for the isolation of mitochondria, however, they are not designed to isolate plasma membrane and the sample yield is significantly less than that from density centrifugation based isolation protocols6,7.
Methods that utilize ultracentrifugation to obtain fractions are more time consuming, but often result in purer fractions than detergent-based kits. To isolate plasma membranes from cells without first solubilizing them (resulting in contamination with membrane organelles) requires them to be lysed by a non-detergent method followed by separation of cellular components via differential centrifugation&#8212;with plasma membrane isolation requiring speeds of 100,000 &#215; g to accomplish. In many cases, differential centrifugation must be followed by isopycnic density gradient centrifugation for further separation of cellular fractions or removal of contaminants. While these methods are thorough and modifiable, drawbacks include cost, time consumption, and the need for an ultracentrifuge for separation of fractions and further purification via density gradient centrifugation. Most high-speed centrifuges are at a cost that is prohibitive for individual investigators and are often shared, core equipment at academic institutions. Thus, ultracentrifuge availability becomes prohibitive in these situations.
In this fractionation protocol we demonstrate the isolation of subcellular fractions without the use of solubilizing detergents and without high speed centrifugation. This method will allow researchers to isolate the plasma membrane, mitochondria and cytoplasmic components of a eukaryotic cell with minimal contamination between fractions.

<table>
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>TCI Chemicals</td>
			<td>D0540</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>VWR</td>
			<td>22877-282</td>
			<td>For Homogenization</td>
		</tr>
		<tr>
			<td>end-over-end rotator</td>
			<td>Barnstead</td>
			<td>N/A</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>Alfa Aesar</td>
			<td>J61721</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>GAPDH (14C10)</td>
			<td>Cell Signalling Technologies</td>
			<td>2118</td>
			<td>For detection of cytoplasmic fractions on western blot, dilution: 1:10000</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>J848</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Alfa Aesar</td>
			<td>12315</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Na, K-ATPase a1 (D4Y7E)</td>
			<td>Cell Signalling Technologies</td>
			<td>23565</td>
			<td>For detection of plasma membrane fractions on western blot, dilution: 1:1000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>793566</td>
			<td>For Lysis buffer A</td>
		</tr>
		<tr>
			<td>phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>VWR</td>
			<td>M145</td>
			<td>For Cytoplasm Extraction and Homogenization Buffer</td>
		</tr>
		<tr>
			<td>probe sonicator</td>
			<td>Qsonica</td>
			<td>Q125-110</td>
			<td>For Final Samples</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, General Use</td>
			<td>VWR</td>
			<td>M221-1ML</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>refrigerated centrifuge</td>
			<td>Beckman-Coulter</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>VWR</td>
			<td>227</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>sodium orthovanadate (SOV)</td>
			<td>Sigma-Aldrich</td>
			<td>450243</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Tris-buffered Saline (TBS)</td>
			<td>VWR</td>
			<td>788</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>VDAC (D73D12)</td>
			<td>Cell Signalling Technologies</td>
			<td>4661</td>
			<td>For detection of mitochondrial fractions on western blot, dilution: 1:1000</td>
		</tr>
	</tbody>
</table>

cell fractionation of u937 cells in the absence of high-speed centrifugation

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.

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...

Watch this Scientific Journal Video about Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation at JoVE.com

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

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.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

This protocol allows researchers to obtain distinct fractions of a cell without the use of specialized equipment, expensive reagents or complex techniques.The primary advantage of this protocol is the simplicity of the techniques which make it modifiable and easy to perform.Demonstrating the procedure will be Matt Deragon, Rob Haluska and Alexa Hodges.Three graduate students from my laboratory.To begin centrifuge the prepared cell suspension to create a pellet.Remove the supernatant and re-suspend the pellet in room temperature PBS.Centrifuge the cell suspension at 400 xg for ten minutes.Then remove the supernatant and re-suspend the cell pellet in ice cold buffer.Prepare buffer A solution according to the text protocol.Centrifuge the cell suspension at 400 xg for ten minutes and remove the supernatant.Re-suspend the pellet in buffer A solution to a final concentration of 200 million cells per ml, and pipette gently to break up any clumps.After this incubate the cell suspension on an end over end rotator at 4

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13.4K Views.  Albany College of Pharmacy and Health Sciences. This protocol allows researchers to obtain distinct fractions of a cell without the use of specialized equipment, expensive reagents or complex techniques.The primary advantage of this protocol is the simplicity of the techniques which make it modifiable and easy to perform.Demonstrating the procedure will be Matt Deragon, Rob Haluska and Alexa Hodges.Three graduate students from my laboratory.To begin centrifuge the prepared cell suspension to create a pellet.Remove the supernatant and re-su...

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

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

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.

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 ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

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.

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 ...

Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been reported to directly bind to a variety of peptides and play both necessary and pathological roles ranging from mediating structure to electron transfer properties to imparting catalytic function. Quantifying the binding affinity and thermodynamics of these Cu(II)-peptide complexes in vitro provides insight into the thermodynamic driving force of binding, potential competitions between different metal ions for the peptide or between different peptides for Cu(II), and the prevalence of the Cu(II)-peptide complex in vivo. However, quantifying the binding thermodynamics can be challenging due to a myriad of factors, including accounting for all competing equilibria within a titration experiment, especially in cases where there are a lack of discrete spectroscopic handles representing the peptide, the d-block metal ion, and their interactions.
Here, a robust set of experiments is provided for the accurate quantification of Cu(II)-peptide thermodynamics. This article focuses on the use of electronic absorption spectroscopy in the presence and absence of chromophoric ligands to provide the needed spectroscopic handle on Cu(II) and the use of label-free isothermal titration calorimetry. In both experimental techniques, a process is described to account for all competing equilibria. While the focus of this article is on Cu(II), the described set of experiments can apply beyond Cu(II)-peptide interactions, and provide a framework for accurate quantification of other metal-peptide systems under physiologically relevant conditions.

Quantifying the Binding Interactions Between CuII and Peptide Residues in the Presence and Absence of Chromophores

This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been ...