The W. M. Keck Foundation generously provided funding for this work. This material is based upon work supported by FAPERJ research grant 100.039/2014 from the government of Rio de Janeiro State - Brazil for R.D.M., Science Without Borders scholarship 88888.075416/2013-00 from the Coordination for the Improvement of Higher Education Personnel, under the government of Brazil, for H.S.S., the National Science Foundation Graduate Research Fellowship under fellowship number 2014171659 for O.S.S., and by CNPq research grant 202011/2012-7 from the government of Brazil for L.H.F.D.V. P.C.H. is a recipient of a Northwestern University&#39;s Chemistry of Life Processes Institute Postdoctoral Fellowship Award.

Note: This video protocol is based on an associated publication9. Specific reagents, material and equipment necessary to perform all the steps described in this protocol are listed in the materials section. The recipes and information for preparation of all required solutions are itemized in Table 1.
1. Pouring of Gradient Tube Gels
<ol>
	<li>Assembling the Casting System
	<ol>
		<li>Using a flame-heated razor blade or scalpel, cut a 20 ml serological pipette along an orthogonal section 10 cm from the top of the dispensing tip. Ensure that the cut surface is smooth. Keep the bottom piece (with the tip) and discard the top piece. Clamp the bottom pipette piece vertically (conical tip facing down) to a support stand. 
		CAUTION: Do not touch the heated blade metal, use proper handles or a plier to avoid burns.</li>
		<li>Cut 3 cm of flexible tubing and connect it to the cut pipet by stretching it over the dispensing tip. Connect the flow control valve at the other end of the tube.</li>
		<li>Link the bottom of the valve to the dispensing tip of the gradient mixer using a piece of tubing. Keep the valve open.</li>
		<li>Set the gradient mixer on top of a magnetic stirrer. Place magnetic bars inside the mixing chambers.</li>
		<li>Position the dispensing tip of the gradient mixer 5-10 cm higher than the top of the pipette piece to allow for the gel to be poured by gravity.</li>
		<li>Test the system for leaks with deionized water. If no leaks are found, dry the system by blowing air inside it before continuing.</li>
		<li>Cover the top of the pipette piece with two or more layers of tape. Puncture a hole through the tape layers in the center of the circular opening. Ensure that the puncture is large enough for the glass tube to pass tightly through.</li>
		<li>Slide the glass tube, where the gel will be cast, through the punctured hole, stretching the tape so the glass fits tightly. Insert the glass tube so that the bottom of the tube is 2 mm above the pipette bottom.</li>
		<li>Check to see if the glass tube is held and centralized and is not touching the pipette wall once the system is prepared. The system is now ready for casting.</li>
	</ol>
	</li>
	<li>Casting Gel
	<ol>
		<li>Prepare gel buffer, APS and Coomassie solutions (solutions 1-3, Table 1).</li>
		<li>Prepare 12% T (solution 4, Table 1) and 1% T (solution 5, Table 1) separating gel solutions. Add 10 &#181;l of Coomassie solution in the 12% T separating gel solution. Add APS and TEMED in both solutions immediately before pouring in gradient mixer. 
		CAUTION: polyacrylamide is highly neurotoxic and gloves must be worn.</li>
		<li>Make sure the dispensing valve of the gradient mixer is closed. Add the 12% T separating gel solution to the mixing chamber farthest from the dispensing tip. Open the valve between both chambers and let the solution flow into the next chamber.</li>
		<li>Close the valve and pipette the dislocated 12% T separating gel solution back to the previous chamber. This is done to avoid air bubbles between chambers.</li>
		<li>Add the 1% T separating gel solution to the mixing chamber closest to the dispensing tip. Turn on the magnetic stirrer.</li>
		<li>Open both gradient mixer valves at the same time to allow the separating gel solutions to be mixed and to flow into the pipette and glass tube.</li>
		<li>Once the separating gel solutions in the gradient mixer are depleted, uncouple the tubing from the mixer and couple it to a 10 ml syringe. Make sure the end of the tube remains above the liquid level in the pipette until the syringe is firmly attached.</li>
		<li>Use the syringe to gently push the remaining separating gel solution from the tubing into the pipette and glass tube. Close the pipette valve before allowing air bubbles into it.</li>
		<li>Gently add a layer of about 0.25 ml of water-saturated butanol (solution 6, Table 1) on top of the gel solution inside the glass tube to seal the polymerization reaction from oxygen in air and to flatten the top meniscus.</li>
		<li>Clean the pouring apparatus immediately with deionized water (do not use detergent).</li>
		<li>Wait overnight for complete gel polymerization at room temperature.</li>
	</ol>
	</li>
	<li>Detaching Gel Tube from Device and Storing
	<ol>
		<li>On the following day, carefully peel the tape off the top of the device.</li>
		<li>Disconnect the valve from the tubing under the pipette. Release the pipette from the clamp and perform steps 1.3.3. and 1.3.4. over a sink.</li>
		<li>Couple a 10 ml syringe filled with deionized water to the flexible tubing. Push water through the tubing into the pipette, slowly sliding the polymerized gel out of the pipette.</li>
		<li>Hold the pipette horizontally and carefully secure the gel sliding out of the open end.</li>
		<li>Cut off excess polyacrylamide beneath and around the glass tube using a razor blade. Make sure to create a smooth end on the gel within the tube, by making it flush with the tip of the glass tube.</li>
		<li>Dispense any remaining butanol from the top side of the gel and set the gel tube in an uncapped 15 ml centrifuge tube. Mix 0.5 ml gel buffer (solution 1, Table 1) with 4.5 ml ultra-pure water and add about 2 ml of the solution to both the top side (within glass tube) and the bottom side (in centrifuge tube) of gel tube.</li>
		<li>Cover the full top side of the centrifuge tube, including the glass tube opening, with parafilm to prevent buffer evaporation. Gels can be stored at 4 &#176;C for about 2 weeks.</li>
	</ol>
	</li>
</ol>
2. Sample Preparation
<ol>
	<li>Weigh two mouse hearts and mince them in a Petri dish using a razor blade.</li>
	<li>Add the processed tissue to a mortar and add sufficient liquid nitrogen to cover the sample. Pulverize the tissue with a pestle, adding more liquid nitrogen when it evaporates.</li>
	<li>Once the tissue is thoroughly ground, let the nitrogen evaporate and add 1 ml of lysis buffer (solution 7, Table 1) at 4 &#176;C for every 100 mg of tissue. Move solution into a centrifuge tube.</li>
	<li>Centrifuge at 2,000 x g for 5 min at 4 &#176;C. Carefully collect the supernatant in a new tube and discard the pellet. Quantify the protein concentration in the supernatant using the bicinchoninic acid method12, or as preferred. Note: Native Cell lysates or subcellular fractions may be prepared as preferred. CN-GELFrEE is compatible with various different native sample preparations.</li>
	<li>Prepare the solubilization buffer solution and loading solution (solutions 8 and 9, Table 1).</li>
	<li>If the sample is a pellet, resuspend it in 50-150 &#181;l of solubilization buffer. Keep the sample on ice for 15 min.</li>
	<li>Buffer exchange the resuspended sample or the cell lysate with solubilization buffer using a 30 kDa-MWCO ultra centrifugal filter.</li>
	<li>Centrifuge the protein sample at 4 &#176;C and 10,000 x g for 5-10 min or until the retained fraction volume is down to approximately 20 &#181;l. Discard the flow-through.</li>
	<li>Add 400 &#181;l of solubilization buffer to the retained fraction and mix by pipetting. Centrifuge at 4 &#176;C and 10,000 x g for 5-10 min or until the retained fraction is down to approximately 20 &#181;l. Discard the flow-through.</li>
	<li>Repeat step 2.9 two additional times.</li>
	<li>Dilute the filtered sample (approximately 20 &#181;l) in 100 &#181;l of solubilization buffer.</li>
	<li>Add 20 &#181;l of loading solution to sample.</li>
</ol>
3. CN-GELFrEE Device
<ol>
	<li>To perform CN-GELFrEE fractionation, manufacture the device parts by referring to a machine shop. Manufacture the device parts in Teflon. Please refer to Figure 1 for all information necessary to manufacture the parts and to Table 2 for the number of necessary parts for one device. 
	Safety Considerations: Wear appropriate personal protective equipment. Care should be taken around the high-voltage power supplies, and all junctions should be correctly mounted. Do not touch the device or any adjacent wet area while voltage is on and maintain the surrounding bench area dry during the electrophoresis. Furthermore, the power supply should be completely turned off when handling the device and/or collecting fractions.</li>
	<li>Prepare the anode buffer and cathode buffer (solutions 10-11, Table 1) and keep them at 4 &#176;C or in ice.</li>
	<li>Fit the top end of the gel tube (end without gel) through a spacer, an O-ring and a second spacer, in a &#34;sandwich&#34; fashion.</li>
	<li>Add the buffer chamber (cathode) after the second spacer, pass the screws fully through the side holes, add and tighten nuts to both sides. Ensure that the top end of the glass tube is fixed before the top aperture of the buffer chamber, not directly under it or past it.</li>
	<li>Fit the bottom end of the gel tube through a spacer, an O-ring, the collection chamber, and a second spacer.</li>
	<li>Add the buffer chamber (anode) after the second spacer, pass the screws fully through the side holes, add and tighten nuts to both sides. Ensure that the bottom end of the gel tube is fixed past the top aperture of the buffer chamber, close but not touching the back wall.</li>
	<li>Set the CN-GELFrEE device vertically with the cathode buffer chamber upwards using a support stand with clamps.</li>
	<li>Add anode buffer to the anode buffer chamber (bottom) and cathode buffer to the cathode buffer chamber (top). Keep all buffers at 4 &#176;C.</li>
	<li>Remove air bubbles from inside the glass tube by gently tapping the glass tube and check the system for leakage.</li>
	<li>Make sure the platinum electrodes are in contact with the buffers in the chambers and that both gel tube ends are immersed in the buffer within each chamber.</li>
	<li>Connect power cords from the power supply to the respective banana plugs: negative on the cathode buffer chamber (top) and positive on the anode buffer chamber (bottom).</li>
	<li>Load the sample using a gel load tip to top surface of the gel, inside the gel tube.</li>
	<li>Run the gel at 1 W constant, to avoid overheating, until the red dye front is at the bottom of the gel tube. 
	CAUTION: Turn off power supply before continuing.</li>
	<li>Discard anode and cathode buffers.</li>
	<li>Untighten the nuts by the anode buffer chamber and disassemble the bottom spacer and the chamber, maintaining a spacer and the O-ring. Place the bottom end of the gel tube inside the collection chamber, before the top aperture, not past or directly under it. Push the spacer and O-ring close to the collection chamber.</li>
	<li>Cut a piece of cellulose dialysis membrane and rehydrate it by immerging in anode buffer. Cover the aperture between the collection chamber and the bottom spacer using the dialysis membrane.</li>
	<li>Assemble the anode buffer chamber after the second spacer, add and tighten the screws and bolts.</li>
	<li>Lay the device horizontally over an ice bucket. Add fresh anode and cathode buffers to the buffer chambers and check the system for leakage.</li>
	<li>Add 150 &#181;l of anode buffer into the collection chamber. Reconnect the power cords to the respective banana plugs.</li>
	<li>Turn on the power supply at 3 mA constant current. The dye front should then start to elute into the buffer in the collection chamber.</li>
	<li>When the entire dye front is eluted, turn off the power supply and collect the first fraction (0 min), transferring it into a low-protein binding microcentrifuge tube.</li>
	<li>Refill the collection chamber with 150 &#181;l of anode buffer. Keep all the collected fractions on ice.</li>
	<li>Collect fractions after the following time intervals from collection of the first fraction: 2 min, 4 min, 6 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min and 60 min. Do not account for time while the power supply is off. Do not forget to turn off the power supply before collecting the fractions and to refill the collection chamber with 150 &#181;l of anode buffer between samples. Turn power supply on again after collection to start eluting the next fraction.</li>
	<li>Dispense the anode and cathode buffers and add fresh solutions to the buffer chambers after 60 min. Collect fractions at 90 min and 120 min.</li>
	<li>Change anode and cathode buffers every hour.</li>
	<li>Run a clear native or a blue native PAGE with the fractions and stain it as preferred. Follow the manufacturer&#39;s instructions or preferred protocol.</li>
	<li>Run a reducing SDS-PAGE and stain it as preferred. Follow the manufacturer&#39;s instructions or preferred protocol.</li>
	<li>Clean up the GELFrEE fractions and submit to native mass spectrometry analyses as described previously9.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53597/53597fig1.jpg" /> 
Figure 1: CN-GELFrEE device. (A) GELFrEE device parts drawings and (B) photograph of manufactured parts; (C) CN-GELFrEE device assembly scheme: (1) buffer chamber, (2) spacer, (3) O-ring, (4) gel tube, (5) collection chamber, and (6) dialysis membrane.
<img alt="Table 1" src="/files/ftp_upload/53597/53597table1.jpg" /> 
Table 1: Table of solutions.
<img alt="Table 2" src="/files/ftp_upload/53597/53597table2.jpg" /> 
Table 2: Table of GELFrEE device parts.

<ol>
	<li>Alberts, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cell+as+a+Collection+of+Protein+Machines:+Preparing+the+Next+Generation+of+Molecular+Biologists.">The Cell as a Collection of Protein Machines: Preparing the Next Generation of Molecular Biologists.</a> Cell. 92 (3), 291-294 (1998).</li><li>Gingras, A. C., Gstaiger, M., Raught, B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+complexes+using+mass+spectrometry.">Analysis of protein complexes using mass spectrometry.</a> Nat. Rev. Mol. Cell Biol. 8 (8), 645-654 (2007).</li><li>Gavin, A. C., B&#246;sche, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415 (6868), 141-147 (2002).</li><li>Puig, O., Caspary, F., 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=The+Tandem+Affinity+Purification+(TAP)+Method:+A+General+Procedure+of+Protein+Complex+Purification.">The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Cremer, K. D., Hulle, M. V., 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=Fractionation+of+vanadium+complexes+in+serum,+packed+cells+and+tissues+of+Wistar+rats+by+means+of+gel+filtration+and+anion-exchange+chromatography.">Fractionation of vanadium complexes in serum, packed cells and tissues of Wistar rats by means of gel filtration and anion-exchange chromatography.</a> JBIC J. Biol. Inorg. Chem. 7 (7-8), 884-890 (2002).</li><li>Tanese, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Scale+Density+Gradient+Sedimentation+to+Separate+and+Analyze+Multiprotein+Complexes.">Small-Scale Density Gradient Sedimentation to Separate and Analyze Multiprotein Complexes.</a> Methods. 12 (3), 224-234 (1997).</li><li>Bunai, K., Yamane, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+and+limitation+of+two-dimensional+gel+electrophoresis+in+bacterial+membrane+protein+proteomics+and+perspectives.">Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives.</a> J. Chromatogr. B. 815 (1-2), 227-236 (2005).</li><li>Wittig, I., Sch&#228;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+limitations+of+clear-native+PAGE.">Advantages and limitations of clear-native PAGE.</a> PROTEOMICS. 5 (17), 4338-4346 (2005).</li><li>Skinner, O. S., Do Vale, L. H. F., 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=Native+GELFrEE:+A+New+Separation+Technique+for+Biomolecular+Assemblies.">Native GELFrEE: A New Separation Technique for Biomolecular Assemblies.</a> Anal. Chem. 87 (5), 3032-3038 (2015).</li><li>Nijtmans, L. G. J., Henderson, N. S., Holt, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+Native+electrophoresis+to+study+mitochondrial+and+other+protein+complexes.">Blue Native electrophoresis to study mitochondrial and other protein complexes.</a> Methods. 26 (4), 327-334 (2002).</li><li>Tran, J. C., Doucette, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel-Eluted+Liquid+Fraction+Entrapment+Electrophoresis:+An+Electrophoretic+Method+for+Broad+Molecular+Weight+Range+Proteome+Separation.">Gel-Eluted Liquid Fraction Entrapment Electrophoresis: An Electrophoretic Method for Broad Molecular Weight Range Proteome Separation.</a> Anal. Chem. 80 (5), 1568-1573 (2008).</li><li>Smith, P. K., Krohn, R. I., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li></ol>

The authors have nothing to disclose.

CN-GELFrEE is derived from the clear native (CN) PAGE buffer system that uses a mixture of anionic and neutral detergents as carrier molecules9 for molecular-weight-based protein fractionation through a gel matrix. The application of CN-GELFrEE to a native mouse heart protein extract generated low complexity fractions with discrete bandwidths while maintaining non-covalent interactions of biomolecular assemblies. Comparison of fractions between CN-PAGE and reducing SDS-PAGE slab gels showed mass shifts that de...

The majority of biological processes happening within a cell are thought to be carried out by protein assemblies rather than single proteins1. Consequently, in order to elucidate the specific biological role of a protein subunit in a cell it is necessary to understand its structural interactions with other proteins or ligands in complexes2. However, studying proteins natively, maintaining their non-covalent interactions and activity, remains challenging. One of the shortcomings of native protein studies is a suitable native separation technique that is compatible with various downstream protein analysis techniques. Therefore, recent interest in separation techniques capable of characterizing non-covalent assemblies of biomolecules has increased sharply3.
Protein separation techniques are imperative to biochemistry, biophysics and various other studies. Current native separation techniques have intrinsic deficiencies that reduce compatibility with downstream analyses, such as low resolution, low throughput, loss to precipitation, and the requirement of large amounts of initial sample. Tandem affinity purification is commonly used for protein interaction studies, but it has to be carried out separately for each protein target, causing it to be incompatible with high-throughput analysis4. Size exclusion chromatography5, selective precipitation with ion affinity chromatography5 and density-gradient separation6 all have provided native separations, but are inherently low-resolution techniques and require high initial sample amounts.
Alternatively, gel-based techniques, such as Blue Native (BN) and Clear Native (CN) PAGE (either 1-D or 2-D), display high-resolution separation. Furthermore, contrasting with other techniques mentioned, both native PAGE separations maintain solubility and native conformation of a broad range of macromolecule species, including hydrophobic proteins. This capability further broadens the proteome coverage reached by these methods7,8 and is achieved through different chemistry between CN and BN-PAGE. CN-PAGE commonly relies on soft charged detergents as carrier molecules, replacing the Coomassie Blue dye in BN-PAGE. BN-PAGE, although associated with higher resolution, has caveats such as reduced enzymatic activity in the separated proteins8 and Coomassie molecule adduct formation, the latter being greatly prejudicial to downstream MS analyses9. Both these methods, however, are traditionally associated with low recovery and narrow proteome coverage due to staining and gel extraction limitations7.
For the study of denatured proteins, there are several techniques that maintain macromolecule solubility while performing high-resolution fractionation with high protein recovery and that are compatible with diverse post-separation protein analysis techniques. Gel-Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE) is one of the fractionation techniques that fit all these characteristics. This method is largely applied in high-throughput top-down proteomics studies, indicating that it is fast and versatile. In GELFrEE, proteins are denatured and separated based on molecular weight through a tubular gel matrix, the porosity of which can be varied based on sample requirements and the desired fractionation results. Fractions are eluted in liquid phase, thus reducing the recovery limitations associated with SDS-PAGE while maintaining high resolution. Fraction aliquots can then be analyzed by 1-D PAGE for molecular-weight bandwidth selection11. There is high demand for the advantages associated with GELFrEE in native separation techniques. The method described herein, Clear Native GELFrEE (CN-GELFrEE), is a native adaptation of GELFrEE. In order to be compatible with a broad spectrum of macromolecules in their native state, this method relies not only on the soft CN-PAGE chemistry, but also on a porosity gradient separation, which reduces protein precipitation by eliminating the harsh transition between porosities present in discontinuous gel systems9. When applied to fractionate protein complexes extracted from mouse heart, eluted fractions displayed high recovery and a high-resolution separation of a wide range of molecular weights was obtained. Further, the resulting fractions are compatible with most downstream biochemical and biophysical protein analyses.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>30% acrylamide/bis solution, 37.5:1</td><td>Bio-Rad</td><td>161-0158</td><td>This solution is neurotoxic.</td></tr><tr><td>6-aminohexanoic acid (&amp;epsilon;-aminocaproic acid)</td><td>Sigma-Aldrich</td><td>A2504</td><td>This chemical is an irritant.</td></tr><tr><td>Ammonium persulfate (APS)</td><td>Fisher Scientific</td><td>7727-54-0</td><td>This chemical is flammable, toxic, and corrosive.</td></tr><tr><td>Coomassie blue G250</td><td>Bio-Rad</td><td>161-0406</td><td></td></tr><tr><td>Dodecylmaltoside</td><td>Sigma-Aldrich</td><td>D4641</td><td>This chemical is an irritant.</td></tr><tr><td>Ethylene glycol tetraacetic acid (EGTA)</td><td>FLUKA</td><td>3777</td><td>This chemical is toxic.</td></tr><tr><td>Ethylenediamine tetraacetic acid (EDTA)</td><td>Fisher Scientific</td><td>BP120</td><td>This chemical is toxic.</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Liquid nitrogen</td><td>Any supplier</td><td>n/a</td><td>Store liquid nitrogen in containers&lt;br/&gt; designed for low-temperature&lt;br/&gt; liquids</td></tr><tr><td>Mouse heart</td><td>Bioreclamation LLC</td><td>MSE-HEART</td><td></td></tr><tr><td>n-butanol</td><td>Fisher Scientific</td><td>71-36-3</td><td>This chemical is flammable and toxic.</td></tr><tr><td>Ponceau S</td><td>Sigma-Aldrich</td><td>BP103</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Thermo Fisher Scientific</td><td>78430</td><td>This chemical is toxic.</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>30970</td><td>This chemical is an irritant.</td></tr><tr><td>Sucrose</td><td>JTBaker</td><td>4097</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Bio-Rad</td><td>161-0800</td><td>This chemical flammable and irritant.</td></tr><tr><td>Tris-HCl</td><td>Amresco</td><td>M151</td><td></td></tr><tr><td>Trycine</td><td>Bio-Rad</td><td>161-071</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gradient mixer</td><td>Hoefer</td><td>SG15</td><td></td></tr><tr><td>Magnetic stir&amp;nbsp;</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>Magnetic bars&amp;nbsp;</td><td>Any supplier</td><td>n/a</td><td>Small enough to fit inside the gradiet mixer chambers.</td></tr><tr><td>Power supply</td><td>Bio-Rad</td><td>164-5056</td><td>Voltage up to 1,500 V</td></tr><tr><td>Refrigerated centrifugue</td><td>Eppendorf</td><td>022622552</td><td>Up to 15,000 x g</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>30 kDa-MWCO ultra centrifugal filter</td><td>Millipore</td><td>UFC503096</td><td></td></tr><tr><td>Flexible tubing</td><td>Saint-Gobain</td><td>B000FOV1J0</td><td>Approximately 1 m length</td></tr><tr><td>Ice bucket</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>Liquid/air metallic or plastic valve. (&lt;em&gt;e.g.&lt;/em&gt; aquarium air/water flow control lever valve)</td><td>Any supplier&amp;nbsp;</td><td>n/a</td><td>Aquarium valves are compatible.</td></tr><tr><td>Mortar and pestle</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>Native PAGE 3-12% T slab gel</td><td>Invitrogen</td><td>BN1003</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>08-757-13</td><td></td></tr><tr><td>Protein low bind tube 1.5&amp;nbsp;ml</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Razor blade</td><td>IDL Tools</td><td>TE05-091C</td><td></td></tr><tr><td>Regenerated cellulose dialysis tubing 3,500 MWCO</td><td>Fisher Scientific</td><td>21-152-9</td><td></td></tr><tr><td>SDS-PAGE slab gel for any kDa</td><td>Bio-Rad</td><td>456-9036</td><td></td></tr><tr><td>Serological pipets (25&amp;nbsp;ml)</td><td>VWR</td><td>89130-900</td><td></td></tr><tr><td>Syringe (10 ml)</td><td>Any supplier</td><td>n/a</td><td></td></tr></tbody></table>

cn-gelfree - clear native gel-eluted liquid fraction entrapment electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

200 &#181;g of proteins extracted from cryogenically ground mouse hearts were fractionated natively into 14 fractions of 150 &#181;l each, using the CN-GELFrEE method in a 1-12% T gel tube. An aliquot of 10 &#181;l of each fraction was run on a CN-PAGE slab gel and silver stained. A clear depiction of the fractionation and resolution obtained with CN-GELFrEE fractionation is shown in Figure 2A. Fractions were collected over two hours and the gel assay shows a consistent p...

Watch this Scientific Journal Video about CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis at JoVE.com

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

cn-gelfree-clear-native-gel-eluted-liquid-fraction-entrapment

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Chemistry

11.5K Views.  Northwestern University. This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Video: CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

Biology

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Biochemistry

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Separating Protein with SDS-PAGE

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions.
This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.

Separating Protein with SDS-PAGE: Procedure and Applications

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their ...

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Basic Biology

Basic Methods in Cellular and Molecular Biology

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
<div class="chapter_text" id="script_0">
Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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<div class="chapter_text" id="script_1">
2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
</div>
<div class="chapter_text" id="script_2">
Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
</div>
<div class="chapter_text" id="script_3">
Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
</div>

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...

Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Source: Zhan, X., et al. <a href="https://www.jove.com/v/56739">Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome</a> J. Vis. Exp. (2018)
In this video, we demonstrate a two-dimensional gel electrophoresis method that separates individual proteins from a complex protein mixture obtained from tumor tissues of human nonfunctional pituitary adenoma (NFPA). The proteins are separated based on their electric charge in the first dimension and further separated based on molecular weights in the second dimension.

Source: Zhan, X., et al. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue ...