This work was supported by NIH R15-HL135675-01 and NIH 2 R15-HL135675-02 to T.J.L.

1. Prepare buffers and reagents
<ol>
	<li>Prepare fresh solutions of phosphatase and protease inhibitors.
	<ol>
		<li>Add 17.4 mg of phenylmethanesulfonyl fluoride (PMSF)to 1 mL of 100% ethanol to prepare a 100 mM stock. 
		&#8203;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.</li>
		<li>According to the manufacturer&#39;s instructions, prepare a commercially available protease inhibitor cocktail (100x).</li>
		<li>Add 91.9 mg of sodium orthovanadate (SOV) to 1 mL of deionized water to prepare a 500 mM stock. 
		NOTE: Wear protective equipment when handling SOV as it is hazardous if ingested, inhaled, or upon contact with eyes. Severe overexposure may result in death.</li>
	</ol>
	</li>
	<li>Prepare lysis buffer A, cytoplasmic isolation (CI) buffer, cell solubilization (CS) buffer, nuclear lysis (NL) buffer, lysis buffer B, cell homogenization (CH) buffer, iodixanol diluent, and detergents.
	<ol>
		<li>Add 8.7 g of sodium chloride (NaCl) and 50 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1 M, pH 7.4) to 950 mL of deionized water to prepare lysis buffer A (final concentrations of 150 mM NaCl and 50 mM HEPES).</li>
		<li>Prepare stock solutions of detergents as follows: add 0.1 g of sodium dodecyl sulfate (SDS) to 10 mL of lysis buffer A (final concentration of 1% SDS), add 0.1 g of sodium deoxycholate to 10 mL of lysis buffer A (final concentration of 1%), add 2.5 mg of digitonin to 10 mL of lysis buffer A (final concentration of 250 &#181;g/mL), and add 1 mL of non-ionic, non-denaturing detergent (see the Table of Materials) to 9 mL of lysis buffer A (final concentration of 10% (v/v)).</li>
		<li>Prepare CI buffer by adding 10 &#181;L of PMSF stock (100 mM), 10 &#181;L of protease inhibitor (100x), 2 &#181;L of SOV stock (500 mM), and 100 &#181;L of stock digitonin (250 &#181;g/mL) to 878 &#181;L of lysis buffer A (final concentrations of 1 mM PMSF, 1x protease inhibitor, 1 mM SOV, and 25 &#181;g/mL digitonin). Keep the solution on ice until addition to cell pellet.</li>
		<li>Prepare CS buffer by adding 10 &#181;L of PMSF stock (100 mM), 10 &#181;L of protease inhibitor (100x), 2 &#181;L of SOV stock (500 mM), 100 &#181;L of non-ionic, non-denaturing detergent stock (see the Table of Materials) (10%), and 118 &#181;L of hexylene glycol stock (8.44 M) to 760 &#181;L of lysis buffer A (final concentrations of 1 mM PMSF, 1x protease inhibitor, 1 mM SOV, 1% non-ionic, non-denaturing detergent, and 1 M hexylene glycol). Keep the solution on ice until addition to cell pellet.</li>
		<li>Prepare NL buffer by adding 10 &#181;L of PMSF stock (100 mM), 10 &#181;L of protease inhibitor (100x), 2 &#181;L of SOV stock (500 mM), 50 &#181;L of sodium deoxycholate stock (10%), 100 &#181;L of SDS stock (1%), and 118 &#181;L of hexylene glycol stock (8.44 M) to 710 &#181;L of lysis buffer A (final concentrations of 1 mM PMSF, 1x protease inhibitor, 1 mM SOV, 0.5% sodium deoxycholate (v/v), 0.1% SDS (w/v), and 1 M hexylene glycol). Keep the solution on ice until addition to nuclear pellet.</li>
		<li>Add 20 mL of HEPES (1 M, pH 7.4), 0.74 g of potassium chloride (KCl), 0.19 g of magnesium chloride (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), 38.3 g of mannitol, and 23.9 g of sucrose to 980 mL of deionized water to prepare lysis buffer B (final concentrations of 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 CH buffer by adding 10 &#181;L of PMSF stock (100 mM) and 2 &#181;L of SOV stock (500 mM) to 988 &#181;L of lysis buffer B (final concentrations of 1 mM PMSF and 1 mM SOV; adjust the final volume to accommodate the number of cells being lysed). Keep the solution on ice until addition to cell pellet.</li>
		<li>Prepare iodixanol diluent by addition of 12 mL of HEPES (1 M, pH 7.4), 447 mg of KCl, 114 g of MgCl2, 1.2 mL of 0.5 M EDTA, 1.2 mL of 0.5 M EGTA, 21.3 g of mannitol, and 14.4 g of sucrose to 88 mL of deionized water (final concentrations of 120 mM HEPES, 60 mM KCl, 12 mM MgCl2, 6 mM EDTA, 6 mM EGTA, 1200 mM mannitol, and 420 mM sucrose).</li>
		<li>Store buffers at 4 &#176;C and digitonin at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Cytosolic protein isolation
NOTE: The following steps will allow for the growth and expansion of U937 cells followed by extraction of cytosolic proteins. At the concentration used, digitonin will permeabilize the plasma membrane without disrupting it, allowing for the release of cytosolic proteins and retention of other cellular proteins.
<ol>
	<li>Culture the cells in RPMI 1640 with 10% fetal bovine serum at 37 &#176;C and 5% CO2. Ensure that the cells are grown to a final total of 6 x 108 cells. 
	NOTE: In this protocol, cells were counted using a standard hemocytometer and microscope.</li>
	<li>Centrifuge the cultured cells at 400 &#215; g for 10 min. After discarding the supernatant, resuspend the cell pellet in room temperature phosphate-buffered saline (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 the cells. Discard the supernatant, and resuspend the cell pellet in ice-cold lysis buffer A (prepared in step 1.2.1) at a final concentration of 2 &#215; 107 cells/mL.</li>
	<li>Remove 7.5 mL of the cells suspended in lysis buffer A (3 &#215; 107 cells), and keep on ice for nuclear protein extraction (section 6). Centrifuge the cell suspension at 4 &#176;C, 400 &#215; g for 10 min to pellet the cells. 
	NOTE: Perform all subsequent steps at 4 &#176;C or on ice, and prechill all buffers.</li>
	<li>Discard the supernatant, resuspend the cell pellet in CI buffer at a final concentration of 2 &#215; 107 cells/mL, and pipette gently to break up clumps. Rotate the cell suspension end-over-end at 4 &#176;C for 20 min.</li>
	<li>Centrifuge the cell suspension at 4 &#176;C, 400 &#215; g for 10 min, and transfer the supernatant to a clean centrifuge tube. Save the cell pellet, and store on ice. Centrifuge the collected supernatant at 4 &#176;C, 18,000 &#215; g for 20 min to pellet cellular debris. 
	NOTE: This high-speed centrifugation step is critical for preventing contamination of the cytosolic fraction with organelle and membrane-bound proteins.</li>
	<li>Discard the pellet after transferring the supernatant to a clean centrifuge tube. Repeat both centrifugation steps in step 2.6 until no pellet is obtained following centrifugation.</li>
	<li>Collect the supernatant, which is the cytosolic fraction. For short-term storage (1 month), ensure that the supernatant is stored at 4 &#176;C. For long-term storage (&#62;1 month), combine the supernatant with 1x Laemmli buffer containing 1x reducing agent, heat at 95 &#176;C for 7 min, and store at -20 or -80&#176;C.</li>
	<li>Resuspend the cell pellet (from step 2.6) in lysis buffer A at a final concentration of 4 &#215; 106 cells/mL; pipette gently to break up clumps.</li>
</ol>
3. Cell homogenization
NOTE: The following steps will allow for the mechanical homogenization of digitonin-treated cells (from step 2.9), which is necessary for the isolation of the mitochondrial and membrane protein fractions.
<ol>
	<li>Centrifuge the cell suspension in lysis buffer A (prepared in step 2.9), save the pellet, and discard the supernatant to remove excess digitonin and cytosolic contaminants from the cell pellet. 
	NOTE: Repeated washes in lysis buffer A may be performed to remove excess cytosolic contaminants.</li>
	<li>Resuspend the cell pellet in ice-cold CH buffer (prepared in step 1.2.7) at a final concentration of 4 &#215; 106 cells/mL. Incubate the cell suspension on ice for 30 min.</li>
	<li>If using a bead-based method for mechanical lysis, place 30 g of prewashed stainless steel 3.2 mm beads into a 50 mL skirted tube, fill with 15 mL of lysis buffer B, and place on ice to chill. If using a Dounce homogenizer (with a tight-fitting B pestle), fill with an appropriate volume of lysis buffer B, insert the pestle, and place on ice to chill.</li>
	<li>Following incubation (step 3.2), discard lysis buffer B from the bead tubes (or Dounce homogenizer), and transfer 15 mL of the cell suspension to the bead tube (or an appropriate volume to the homogenizer).</li>
	<li>If using the bead-based method, place the skirted bead tubes containing the cell suspension in the blender device, and configure to run for 5 min at speed 8. If using a Dounce homogenizer, keep it on ice, and perform 40 passes with the pestle using slow, even strokes (or utilize an alternative method of mechanical cell lysis as detailed in the discussion section). 
	NOTE: If using a blender device different from the one used here, speed and time may need to be determined empirically.</li>
	<li>Transfer the homogenate to a clean centrifuge tube, and centrifuge it at 400 &#215; g for 10 min at 4 &#176;C. Transfer the supernatant to a clean centrifuge tube and save; discard the pellet. 
	&#8203;NOTE: The protocol may be paused here and the homogenate stored at 4 &#176;C for the short term (24 h).</li>
</ol>
4. Debris removal and isolation of crude mitochondrial and membrane fractions
NOTE: The following steps will allow for the removal of cellular debris by centrifuging the homogenate at increasing speeds. This is followed by differential centrifugation for the isolation of crude mitochondrial and membrane fractions.
<ol>
	<li>Centrifuge the homogenate (from step 3.6) at 500 &#215; g for 10 min at 4 &#176;C. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.</li>
	<li>Centrifuge the supernatant (from step 4.1) at 1,000 &#215; g for 10 min at 4 &#176;C. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.</li>
	<li>Centrifuge the supernatant (from step 4.2) at 2,000 &#215; g for 10 min at 4 &#176;C. Transfer the supernatant to a clean centrifuge tube, and discard any pellet.</li>
	<li>Centrifuge the supernatant (from step 4.3) at 4,000 &#215; g for 20 min at 4 &#176;C. Transfer the supernatant to a clean centrifuge tube, and save the pellet, which is the crude mitochondrial fraction.</li>
	<li>Centrifuge the supernatant (from step 4.4) at 18,000 &#215; g for 1 h at 4 &#176;C. Discard the supernatant, and save the pellet, which is the crude membrane fraction. 
	&#8203;NOTE: The protocol may be paused here, and the pelleted samples stored at 4 &#176;C for the short-term (24 h).</li>
</ol>
5. Isopycnic density gradient purification
NOTE: The following steps utilize isopycnic density gradient centrifugation to purify the crude mitochondrial and membrane fractions.
<ol>
	<li>Prepare a 50% (v/v) working solution of iodixanol by mixing 1 part diluent (prepared in step 1.1.3) to 5 parts iodixanol (60% stock solution (w/v)). Prepare 10%, 15%, 20%, 25%, 30%, and 35% iodixanol solutions (v/v) by mixing 50% working solution (v/v) with lysis buffer B in appropriate quantities.</li>
	<li>Resuspend the crude mitochondrial and membrane pellets (from steps 4.4 and 4.5) each in 200 &#181;L of lysis buffer B. Adjust to 45% iodixanol (v/v) by adding 1800 &#181;L of working iodixanol solution (50% (v/v)) to 200 &#181;L of the resuspended pellet.</li>
	<li>Create an iodixanol discontinuous gradient as follows (adjust the volumes as appropriate for the capacity of the ultracentrifuge tube): Add 1 mL of 15% iodixanol (v/v) to the bottom of an 8 mL, open-top, thin-walled ultracentrifuge tube, place 1 mL of 20% iodixanol (v/v) below the first layer (by the underlaying technique), followed by underlaying 1 mL of 25%, 1 mL of 30%, and 1 mL of 35% iodixanol (v/v). 
	NOTE: There should be 2 gradients created at this step, 1 for the mitochondrial fraction and 1 for the membrane fraction.</li>
	<li>In 1 gradient, add the 2 mL of the crude mitochondrial pellet (suspended in 45% iodixanol (v/v)) using the underlay technique to the bottom of the tube below the 35% iodixanol (v/v). In the other gradient, add the 2 mL of the crude membrane pellet (suspended in 45% iodixanol (v/v)) using the underlay technique to the bottom of the tube below the 35% iodixanol (v/v).</li>
	<li>Add 1 mL of 10% iodixanol (v/v) to the top of each gradient tube by overlaying it on top of the 15% layer. Balance the tubes within 0.1 g of each other by the addition of 10% iodixanol (v/v).</li>
	<li>Spin the density gradient tubes at 4 &#176;C for 18 h at 100,000 &#215; g. Be sure to set acceleration and deceleration to the minimum values for the ultracentrifuge being used. 
	NOTE: In the mitochondrial gradient, there will be a visible band at the interface between 25% and 30% iodixanol (v/v). This is the pure mitochondrial fraction. In the membrane gradient, there will be a visible band in the 15% iodixanol (v/v) fraction. This is the pure membrane fraction. There may be additional bands in the 25% and 30% iodixanol (v/v) fractions in the membrane gradient. This is mitochondrial contamination.</li>
	<li>Collect fractions from the top of the tube in 1 mL aliquots, being careful to minimize the volume of the fraction containing the visible bands and changing the pipette tip after each layer is collected. Alternatively, puncture the side of the thin-walled tube with a needle, and collect the visible bands.</li>
	<li>Store the samples as described in step 2.8 until verification by protein assay and western blot.</li>
</ol>
6. Nuclear protein isolation
NOTE: Using ionic and non-ionic detergents as well as techniques such as sonication and centrifugation, the following steps will solubilize all cellular membranes and allow for the isolation of nuclear proteins.
<ol>
	<li>Centrifuge the 7.5 mL aliquot of cells suspended in lysis buffer A (from step 2.4), and discard the supernatant. Add 800 &#181;L of ice-cold CS buffer (prepared in step 1.2.4), and resuspend the pellet by pipetting and vortexing. Incubate the samples on ice (or at 4 &#176;C) for 30 min to disrupt the plasma and organelle membranes while protecting the nuclear proteins.</li>
	<li>Centrifuge the cell suspension at 7,000 &#215; g for 10 min at 4 &#176;C, and discard the supernatant. Add 800 &#181;L of ice-cold NL buffer (prepared in step 1.2.5) to the nuclear pellet after inclusion of 1U/&#181;L benzonase. Resuspend the pellet by gently pipetting. Incubate on an end-over-end rotator for 30 min at 4 &#176;C to disrupt the nuclear membrane.</li>
	<li>Sonicate for 5 s at 20% power with sample tubes cooled in an ice bath (3x, with 5 s pauses in between pulses), which along with benzonase (added in step 6.2), will shear the nucleic acids.</li>
	<li>Centrifuge the sonicate at 7,800 &#215; g for 10 min at 4 &#176;C. Collect and save the supernatant, which is the nuclear fraction. Be sure to not disturb the insoluble pellet while collecting the supernatant. For storage, samples can be prepared as described in step 2.8.</li>
</ol>
7. Protein quantification and western blot analysis
NOTE: The following steps will quantify total protein in each fraction and confirm the purity of the subcellular fractions.
<ol>
	<li>Perform a standard Bradford assay to quantify total protein in each fraction. Refer to Table 1 for expected protein yields. 
	NOTE: Other protein assays such as bicinchoninic acid (BCA) assay or absorbance at 280 nm may be used to measure total protein in place of a Bradford assay.</li>
	<li>Combine fractions with 1x Laemmli buffer containing 1x reducing agent, and heat at 95 &#176;C for 7 min. Load 10 &#181;g of each fraction into a standard SDS-polyacrylamide gel electrophoresis (PAGE) gel, and run the gel at 20 mA for 1 h.</li>
	<li>Transfer the resolved proteins to a polyvinyldifluoride (PVDF) membrane by performing a standard immunoblot transfer at 100 V for 30 min. Block the PVDF membrane with 5% milk in 1x Tris-buffered saline (TBS) containing 0.1% of a non-ionic detergent (v/v) for 30 min at room temperature.</li>
	<li>Add the appropriate primary antibodies to detect housekeeping proteins specific to each subcellular fraction, and incubate overnight at 4 &#176;C. Wash the membrane with 5% milk in 1x TBS containing 0.1% of the non-ionic detergent (v/v) for 10 min at room temperature. 
	NOTE: For this protocol, antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10000), histone H3 (1:2000), voltage-dependent anion channel (VDAC, 1:1000), and Na,K+-ATPase were used for cytosolic, nuclear, mitochondrial, and membrane fractions, respectively.</li>
	<li>Add the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies to the membrane (dilute according to the manufacturer&#39;s instructions), and incubate at room temperature for 1 h. Wash the membrane with 5% milk in 1x TBS containing 0.1% of the non-ionic detergent (v/v) for 10 min at room temperature. Develop the blot using standard chemiluminescence.</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, 440-445 (2015).</li><li>Il 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>Clayton, D. A., Shadel, G. S. <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+mitochondria+from+cells+and+tissues.">Isolation of mitochondria from cells and tissues.</a> Cold Spring Harbor Protocols. 2014 (10), 1040-1041 (2014).</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>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>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), 541-566 (2000).</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 of the United States of the America. 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>Bradbury, E. M., Cary, P. D., Crane-Robinson, C., Rattle, H. W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformations+and+interactions+of+histones+and+their+role+in+chromosome+structure.">Conformations and interactions of histones and their role in chromosome structure.</a> Annals of the New York Academy of Sciences. 222, 266-289 (1973).</li><li>McCaig, W. D., Deragon, M. A., Haluska, R. J., Hodges, A. L., Patel, P. S., LaRocca, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fractionation+of+U937+cells+in+the+absence+of+high-speed+centrifugation.">Cell fractionation of U937 cells in the absence of high-speed centrifugation.</a> Journal of Visualized Experiments. (143), e59022 (2019).</li><li>McCaig, W. D., 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></ol>

The authors declare no conflict of interest.

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<sup ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.
The Authors section was updated from:
William McCaig1 
Timothy LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences
to:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Keven J. Hughes1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.

Erratum: Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.
The Authors section was updated from:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Keven J. Hughes1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences
to:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

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-based1,2, relying on the use of buffers containing increasing amounts of detergent to solubilize different cellular components. While this method is rapid and convenient, it results in impure fractions. These are designed to allow researchers to easily isolate one or two components of the cell, but are not complex enough to isolate multiple subcellular fractions from a sample at the same time. Relying solely on detergents usually results in membrane-enclosed organelles and the plasma membrane being indiscriminately solubilized, making separation of these components difficult. An additional complication from the use of these kits is the inability of researchers to alter/optimize them for specific applications, as most of the components are proprietary formulations. Finally, these kits can be prohibitively expensive, with limitations in the number of uses that make them less than ideal for larger samples. 
Despite the availability of kits for the isolation of mitochondria that do not rely on detergents, they are not designed to isolate the plasma membrane and yield significantly lower amounts of sample than standard isolation protocols3,4. While differential centrifugation methods are more time-consuming, they often result in distinct fractions that cannot be obtained with exclusively detergent-based kits1. Separation without the sole use of solubilizing detergents also allows further purification using ultracentrifugation and isopycnic density gradients, resulting in less cross-contamination. This fractionation protocol demonstrates the isolation of subcellular fractions from U937 monocytes using a combination of detergent- and high-speed centrifugation-based approaches. This method will facilitate the isolation of the nuclear, cytoplasmic, mitochondrial, and plasma membrane components of a mammalian cell with minimal contamination between the fractions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender Tissue Homogenizer</td>
			<td>Next Advance</td>
			<td>61-BB50-DX</td>
			<td></td>
		</tr>
		<tr>
			<td>digitonin</td>
			<td>Sigma</td>
			<td>D141</td>
			<td></td>
		</tr>
		<tr>
			<td>end-over-end rotator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH (14C10)&#160;</td>
			<td>Cell Signalling Technologies</td>
			<td>2118</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>97064-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexylene glycol</td>
			<td>Sigma</td>
			<td>68340</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal</td>
			<td>Sigma</td>
			<td>I7771</td>
			<td>Non-ionic, non-denaturing detergent</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M9647</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Na, K-ATPase a1 (D4Y7E)</td>
			<td>Cell Signalling Technologies</td>
			<td>23565</td>
			<td></td>
		</tr>
		<tr>
			<td>Open-Top Polyclear Tubes, 16 x 52 mm</td>
			<td>Seton Scientific</td>
			<td>7048</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep (Iodixanol)&#160;Density Gradient Medium</td>
			<td>Sigma</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, General Use</td>
			<td>VWR</td>
			<td>M221-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>refrigerated centrifuge</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S50-ST Swinging Bucket Rotor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium orthovanadate (SOV)</td>
			<td>Sigma</td>
			<td>567540</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall MX120 Plus Micro-Ultracentrifuge</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Beads 3.2 mm</td>
			<td>Next Advance</td>
			<td>SSB32</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered Saline (TBS)</td>
			<td>VWR</td>
			<td>97062-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td></td>
			<td></td>
			<td>non-ionic detergent in western blotting buffers</td>
		</tr>
		<tr>
			<td>VDAC (D73D12)</td>
			<td>Cell Signalling Technologies</td>
			<td>4661</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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</su...

Watch this Scientific Journal Video about Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification at JoVE.com

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

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 ...

cell-fractionation-u937-cells-isopycnic-density-gradient

Research

JoVE Journal

Biochemistry

3.7K Views.  Albany College of Pharmacy and Health Sciences. 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.

Video: Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

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 ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

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 ...

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.

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. ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

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 ...