Name: cn-gelfree - clear native gel-eluted liquid fraction entrapment electrophoresis
Uploaded: 2016-02-29T14:00:00
Description: Watch this Scientific Journal Video about CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis at JoVE.com

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>

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.

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

Research

JoVE Journal

Chemistry

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...