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 alon...

<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 border="0" cellpadding="0" cellspacing="0" width="820">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:820px;">Reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">30% acrylamide/bis solution, 37.5:1</td>
			<td style="width:176px;">Bio-Rad</td>
			<td style="width:119px;">161-0158</td>
			<td style="width:249px;">This solution is neurotoxic.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">6-aminohexanoic acid (&#949;-aminocaproic acid)</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2504</td>
			<td style="width:249px;">This chemical is an irritant.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Ammonium persulfate (APS)</td>
			<td style="width:176px;">Fisher Scientific</td>
			<td style="width:119px;">7727-54-0</td>
			<td style="width:249px;">This chemical is flammable, toxic, and corrosive.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Coomassie blue G250</td>
			<td style="width:176px;">Bio-Rad</td>
			<td style="width:119px;">161-0406</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Dodecylmaltoside</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4641</td>
			<td style="width:249px;">This chemical is an irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ethylene glycol tetraacetic acid (EGTA)</td>
			<td style="width:176px;">FLUKA</td>
			<td style="width:119px;">3777</td>
			<td style="width:249px;">This chemical is toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ethylenediamine tetraacetic acid (EDTA)</td>
			<td style="width:176px;">Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td style="width:249px;">This chemical is toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Glycerol</td>
			<td style="width:176px;">Fisher Scientific</td>
			<td style="width:119px;">56-81-5</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Imidazole</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:119px;">I2399</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:276px;">Liquid nitrogen</td>
			<td style="width:176px;">Any supplier</td>
			<td style="width:119px;">n/a</td>
			<td style="width:249px;">Store liquid nitrogen in containers 
			designed for low-temperature 
			liquids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Mouse heart</td>
			<td style="width:176px;">Bioreclamation LLC</td>
			<td style="width:119px;">MSE-HEART</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">n-butanol</td>
			<td style="width:176px;">Fisher Scientific</td>
			<td style="width:119px;">71-36-3</td>
			<td style="width:249px;">This chemical is flammable and toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ponceau S</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:119px;">BP103</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Protease Inhibitor Cocktail</td>
			<td style="width:176px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">78430</td>
			<td style="width:249px;">This chemical is toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sodium deoxycholate</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:119px;">30970</td>
			<td style="width:249px;">This chemical is an irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sucrose</td>
			<td style="width:176px;">JTBaker</td>
			<td style="width:119px;">4097</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Tetramethylethylenediamine (TEMED)</td>
			<td style="width:176px;">Bio-Rad</td>
			<td style="width:119px;">161-0800</td>
			<td style="width:249px;">This chemical flammable and irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Tris-HCl</td>
			<td style="width:176px;">Amresco</td>
			<td style="width:119px;">M151</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Trycine</td>
			<td style="width:176px;">Bio-Rad</td>
			<td style="width:119px;">161-071</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Name</td>
			<td style="width:176px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:820px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Gradient mixer</td>
			<td style="width:176px;">Hoefer</td>
			<td style="width:119px;">SG15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Magnetic stir&#160;</td>
			<td style="width:176px;">Any supplier</td>
			<td style="width:119px;">n/a</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Magnetic bars&#160;</td>
			<td style="width:176px;">Any supplier</td>
			<td style="width:119px;">n/a</td>
			<td style="width:249px;">Small enough to fit inside the gradiet mixer chambers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Power supply</td>
			<td style="width:176px;">Bio-Rad</td>
			<td style="width:119px;">164-5056</td>
			<td>Voltage up to 1,500 V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Refrigerated centrifugue</td>
			<td style="width:176px;">Eppendorf</td>
			<td style="width:119px;">022622552</td>
			<td>Up to 15,000 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Name</td>
			<td style="width:176px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:820px;">Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">15 mL conical tube</td>
			<td style="width:176px;">Any supplier</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">30-kDa-MWCO ultra centrifugal filter</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">UFC503096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Flexible tubing</td>
			<td style="width:176px;">Saint-Gobain</td>
			<td>B000FOV1J0</td>
			<td>Approximately 1 m length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ice bucket</td>
			<td style="width:176px;">Any supplier</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:276px;">Liquid/air metallic or plastic valve. (e.g. aquarium air/water flow control lever valve)</td>
			<td style="width:176px;">Any supplier&#160;</td>
			<td style="width:119px;">n/a</td>
			<td style="width:249px;">Aquarium valves are compatible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Mortar and pestle</td>
			<td style="width:176px;">Any supplier</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Native PAGE 3-12% T slab gel</td>
			<td style="width:176px;">Invitrogen</td>
			<td>BN1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Parafilm</td>
			<td style="width:176px;">Bemis</td>
			<td style="width:119px;">PM-996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Petri dish</td>
			<td style="width:176px;">Fisher Scientific</td>
			<td style="width:119px;">08-757-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Protein low bind tube 1.5 mL</td>
			<td style="width:176px;">Eppendorf</td>
			<td>022431081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Razor blade</td>
			<td style="width:176px;">IDL Tools</td>
			<td style="width:119px;">TE05-091C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Regenerated cellulose dialysis tubing 3,500 MWCO</td>
			<td style="width:176px;">Fisher Scientific</td>
			<td style="width:119px;">21-152-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">SDS-PAGE slab gel for any kDa</td>
			<td style="width:176px;">Bio-Rad</td>
			<td style="width:119px;">456-9036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Serological pipets (25 ml)</td>
			<td style="width:176px;">VWR</td>
			<td style="width:119px;">89130-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Syringe (10 ml)</td>
			<td style="width:176px;">Any supplier</td>
			<td style="width:119px;">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 ...

The overall goal of this novel separations technique is to fractionate native samples with high recovery and resolution while maintaining non-covalent macromolecular interactions such as protein complexes. This method can help answer key questions in the study of native macromolecular complexes, such as complex composition and stoichiometry. The main advantage of this technique is its high recovery and low sample dilution, unrivaled by any other native separations method.Begin with assembling the casting system. The first step is to use a hot blade to cut the tip off a 20 milliliter serological pipette. Cut orthogonally 10 centimeters from the top of the dispensing tip.Then, clamp the cut pipette tip down to a support stand. Next, cut a three centimeter length of tubing to attach to the tip. Stretch one end of the tubing over the tip, and attach a flow control valve.Connect the valve to the dispensing tip of a gradient mixer using another piece of tubing. Keep the valve open for now. Next, put the gradient mixer on a magnetic stirrer and load stir bars into the mixing chambers.Now, text the system for leaks using de-ionized water. Then, dry the system by blowing air through it. Cover the top of the pipette section with two layers of tape.Then, puncture a hole through the tape layers, and slide a glass tube through the hole. Extend the glass tube to within two millimeters of the bottom. The tube must fit the tape snugly and not touch the pipette walls.Now, cast the gel. Close the dispensing valve, and add the 12%T separating gel solution to the mixing chamber furthest from the dispensing tip. So bubbles don't form, open the valve between the chambers to let the solution flow.Then, close the valve and transfer the dislocated gel solution back to the previous chamber using a pipette. Now, add 1%T gel to the mixing chamber closest to the dispensing tip, and turn on the magnetic stirrer. Now, open the flow valve, then both gradient mixer valves at the same time to allow the two gel solutions to mix and flow into the glass tube.When finished, attach the tubing to a 10 milliliter syringe, keeping it above the liquid level. Then, use the syringe to push the remaining gel solution from the tubing into the pipette and tube. Before any air gets in, close the valve.Next, gently pipette about 0.25 milliliters of water saturated butanol over the gel to seal the polymerization reaction. Let the reaction go overnight at room temperature. On the following day, carefully peel the tape off the top of the device, disconnect the valve from the tubing under the pipette, and release the pipette from the clamp.Over a sink, attach a 10 milliliter syringe filled with de-ionized water to the tubing, and use this water to slowly force the polymerized gel out of the pipette. Then, trim the excess gel so it is smooth and flush with the glass edge. Store the gel tube in 0.1 X buffer solution, and cover it with parafilm wrap.After manufacturing the parts for the CN-GELFrEE device, assemble them. First, fit the end of the gel tube without gel through a spacer, an O ring, and a second spacer in a sandwich fashion. Then, add the cathode buffer chamber after the second spacer.Pass the screws fully through the side holes and tighten the nuts to both sides. Make certain that the top end of the glass tube is fixed before the top aperture of the buffer chamber and not directly under it or past it. Proceed by fitting the bottom end of the gel tube through a spacer, an O ring, the collection chamber, a second spacer, and then attach the anode buffer chamber like the cathode buffer chamber.Make sure that the bottom end of the gel tube is fixed past the top aperture of the buffer chamber. It should also be close to the back wall without touching it. Now, clamp the electrophoresis device vertically, with the cathode up.Then, load the two chambers with either four degree Celsius anode or cathode buffer. Tap out any air bubbles, and check for leaks. The platinum electrode must touch the buffers, and both gel tube ends must be immersed in buffer.The last step is to connect the gel to the power supply. Load the sample on the top surface of the gel using the load tip. Next, run the gel at one watt until the red dye front has passed to the bottom of the gel.Then, firstly, turn off the power supply, and secondly, discard the buffers. Now, disassemble the bottom spacer and chamber, keeping one spacer and the O ring attached. Put the bottom end of the gel tube into the collection chamber before the top aperture but not past it or directly under it, and push the spacer and O ring close to the collection chamber.Then, cover the aperture with a membrane rehydrated in anode buffer. Assemble the anode buffer chamber after the second spacer. Lay the device horizontally over an ice bucket.Then, refill the buffer chambers, check for leaks, and then add 150 microliters of anode buffer into the collection chamber. Now, send a constant three milliamps to the device. Once the dye front dilutes, turn off the power.Then, collect the first fraction. Transfer it to a low protein binding microcentrifuge tube, and put it on ice. Next, refill the collection chamber with another 150 microliters of anode buffer, and collect the next fraction.Carefully proceed by repeating this process to collect all the fractions. With practice, no protein loss should occur. Every 60 minutes during the fractionation, change the anode and cathode buffer.Proteins were extracted from frozen mouse heart and fractionated natively using CN-GELFrEE in a one to 12 percent T gel tube. Fraction aliquots were run on a CN-PAGE slab gel and silver stained. Each native fraction was subsequently reduced and denatured.Samples were run again on an SDS-PAGE slab gel. Mass shifts and an increase in the number of species were observed in all fractions compared to the native fractions, indicating that the native fraction contained intact protein complexes. The native fractions were then cleaned and analyzed with mass spectrometry.One intact protein complex observed, for example, was homodimeric malate dehydrogenase. It had a charge distribution between 16 plus and 20 plus, and a molecular mass of 72, 843 daltons. The 18 plus charge state was isolated and collisionally activated, resulting in the ejection of monomeric malate dehydrogenase with a molecular mass of 36, 421 daltons.Source activation was applied to the intact complex in order to induce monomer ejection. This allowed isolation, further fragmentation, and identification of the monomers. Thus, CN-GELFrEE is compatible with mass spectrometry and it does not disrupt non-covalent protein-protein interactions of macromolecular assemblies.This procedure generates liquid entrapped native fractions that are likely to be compatible with many downstream analytical methods besides mass spectrometry, including activity assays, binding assays, and microscopy. Don't forget that working with liquid fractions and electricity can be extremely hazardous, and that the electric source should always be off during the handling of liquids or of the device throughout this procedure.

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JoVE Journal

Chemistry

11.2K Görüntüleme.  Northwestern University. The overall goal of this novel separations technique is to fractionate native samples with high recovery and resolution while maintaining non-covalent macromolecular interactions such as protein complexes. This method can help answer key questions in the study of native macromolecular complexes, such as complex composition and stoichiometry. The main advantage of this technique is its high recovery and low sample dilution, unrivaled by any other native separations method.Begin with ass...