Name: mucin agarose gel electrophoresis: western blotting for high-molecular-weight glycoproteins
Uploaded: 2016-06-14T15:00:00
Description: Watch this Scientific Journal Video about Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins at JoVE.com

The authors would like to acknowledge Dr. John Sheehan and Dr. Lubna Abdullah for their guidance and mentoring that were central in the completion of this work. This work was supported by funds from the National Institutes of Health (P01HL108808, UH2HL123645) and the Cystic Fibrosis Foundation Therapeutics, Inc. (EHRE07XX0). Kathryn Ramsey is supported by an NHMRC Early Career Research fellowship.

1. Prepare Buffers for Mucin Gel Western Blotting
<ol>
	<li>Prepare 1 liter of 50x TAE (Tris-acetate-EDTA) buffer.
	<ol>
		<li>In 700 ml of distilled water (dH2O) add 242 g of Tris base (0.4 M), 57.1 g of glacial acetic acid (weigh liquid) (0.2 M) and 14.61 g of ethylenediaminetetraacetic acid (EDTA) (50 mM).</li>
		<li>Adjust pH to 8.0 and make the volume up to 1 liter with dH2O.</li>
	</ol>
	</li>
	<li>Prepare 10 ml of 10x loading buffer.
	<ol>
		<li>Prepare 5 ml of 1x TAE buffer. To do this, ad...

<ol>
	<li>Henderson, A. G., 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=Cystic+fibrosis+airway+secretions+exhibit+mucin+hyperconcentration+and+increased+osmotic+pressure.">Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure.</a> J Clin Invest. 124 (7), 3047-3060 (2014).</li><li>Preciado, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC5B+Is+the+predominant+mucin+glycoprotein+in+chronic+otitis+media+fluid.">MUC5B Is the predominant mucin glycoprotein in chronic otitis media fluid.</a> Pediatr Res. 68 (3), 231-236 (2010).</li><li>Wang, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgG+in+cervicovaginal+mucus+traps+HSV+and+prevents+vaginal+herpes+infections.">IgG in cervicovaginal mucus traps HSV and prevents vaginal herpes infections.</a> Mucosal Immunol. 7 (5), 1036-1044 (2014).</li><li>Linden, S. K., Sutton, P., Karlsson, N. G., Korolik, V., McGuckin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins+in+the+mucosal+barrier+to+infection.">Mucins in the mucosal barrier to infection.</a> Mucosal Immunol. 1 (3), 183-197 (2008).</li><li>Thornton, D. J., 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=Mucus+glycoproteins+from+'normal'+human+tracheobronchial+secretion.">Mucus glycoproteins from 'normal' human tracheobronchial secretion.</a> Biochem J. 265 (1), 179-186 (1990).</li><li>Kesimer, M., Makhov, A. M., Griffith, J. D., Verdugo, P., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpacking+a+gel-forming+mucin:+a+view+of+MUC5B+organization+after+granular+release.">Unpacking a gel-forming mucin: a view of MUC5B organization after granular release.</a> Am J Physiol Lung Cell Mol Physiol. 298 (1), L15-L22 (2010).</li><li>Kesimer, M., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+analysis+of+mucin+core+proteins.">Mass spectrometric analysis of mucin core proteins.</a> Methods Mol Biol. 842, 67-79 (2012).</li><li>van der Post, S., Thomsson, K. A., Hansson, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+enzyme+approach+for+the+characterization+of+glycan+modifications+on+the+C-terminus+of+the+intestinal+MUC2mucin.">Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.</a> J Proteome Res. 13 (12), 6013-6023 (2014).</li><li>Thornton, D. J., Carlstedt, I., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+glycoproteins+on+nitrocellulose+membranes+and+gels.">Identification of glycoproteins on nitrocellulose membranes and gels.</a> Methods Mol Biol. 32, 119-128 (1994).</li><li>Thornton, D. J., Carlstedt, I., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+glycoproteins+on+nitrocellulose+membranes+and+gels.">Identification of glycoproteins on nitrocellulose membranes and gels.</a> Mol Biotechnol. 5 (2), 171-176 (1996).</li><li>Sheehan, J. K., 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=Physical+characterization+of+the+MUC5AC+mucin:+a+highly+oligomeric+glycoprotein+whether+isolated+from+cell+culture+or+in+vivo+from+respiratory+mucous+secretions.">Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions.</a> Biochem J. 347 Pt 1, 37-44 (2000).</li><li>Thornton, D. J., Howard, M., Khan, N., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+glycoforms+of+the+MUC5B+mucin+in+human+respiratory+mucus.+Evidence+for+a+cysteine-rich+sequence+repeated+within+the+molecule.">Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule.</a> J Biol Chem. 272 (14), 9561-9566 (1997).</li><li>Sheehan, J. K., 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=Identification+of+molecular+intermediates+in+the+assembly+pathway+of+the+MUC5AC+mucin.">Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin.</a> J Biol Chem. 279 (15), 15698-15705 (2004).</li><li>Ehre, C., 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=Overexpressing+mouse+model+demonstrates+the+protective+role+of+Muc5ac+in+the+lungs.">Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs.</a> Proc Natl Acad Sci U S A. 109 (41), 16528-16533 (2012).</li><li>Livraghi, A., 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=Airway+and+lung+pathology+due+to+mucosal+surface+dehydration+in+{beta}-epithelial+Na++channel-overexpressing+mice:+role+of+TNF-{alpha}+and+IL-4R{alpha}+signaling,+influence+of+neonatal+development,+and+limited+efficacy+of+glucocorticoid+treatment.">Airway and lung pathology due to mucosal surface dehydration in {beta}-epithelial Na+ channel-overexpressing mice: role of TNF-{alpha} and IL-4R{alpha} signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment.</a> J Immunol. 182 (7), 4357-4367 (2009).</li><li>Martino, M. B., 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+ER+stress+transducer+IRE1beta+is+required+for+airway+epithelial+mucin+production.">The ER stress transducer IRE1beta is required for airway epithelial mucin production.</a> Mucosal Immunol. 6 (3), 639-654 (2013).</li><li>Nguyen, L. P., 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=Chronic+exposure+to+beta-blockers+attenuates+inflammation+and+mucin+content+in+a+murine+asthma+model.">Chronic exposure to beta-blockers attenuates inflammation and mucin content in a murine asthma model.</a> Am J Respir Cell Mol Biol. 38 (3), 256-262 (2008).</li><li>Papakonstantinou, E., 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=COPD+exacerbations+are+associated+with+pro-inflammatory+degradation+of+hyaluronic+acid.">COPD exacerbations are associated with pro-inflammatory degradation of hyaluronic acid.</a> Chest. , (2015).</li><li>Abdullah, L. H., Wolber, C., Kesimer, M., Sheehan, J. K., Davis, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+mucin+secretion+from+human+bronchial+epithelial+cell+primary+cultures.">Studying mucin secretion from human bronchial epithelial cell primary cultures.</a> Methods Mol Biol. 842, 259-277 (2012).</li></ol>

The protocol of mucin Western blotting described in this video combines conventional techniques used in molecular biology to separate and transfer large macromolecules, such as DNA, with regular techniques for protein detection, i.e., immunoblotting. The same technique could be applied to study the biology of complex glycosaminoglycans, such as the breakdown of high-molecular-weight hyaluronic acid18. Although this technique could be used in a broad range of assays, successful agarose Western blotting...

Mucins are normally produced by mucosal surfaces that line cavities exposed to the external environment (e.g., respiratory, digestive, reproductive tracts, ocular surface) as well as internal organs (e.g., pancreas, gallbladder, mammary glands). The presence of these glycoproteins maintains surface hydration and forms a physical barrier against pathogens. Although mucin production is essential to mucosal health, mucin hyperconcentration and/or aberrant mucus properties can lead to duct obstruction, bacterial colonization and chronic inflammation, which can cause irreversible tissue damage. A similar cascade of events are observed in several diseases, e.g., cystic fibrosis1, chronic otitis media2 and cervicovaginal infection3. Therefore, it is important to understand the role of mucins in health and disease and to establish routine protocols for protein identification.
To date, 19 mucins genes have been identified and encode for large polypeptide chains ranging from 1,200 (e.g., MUC1) to 22,000 (e.g., MUC16) amino acids. The mucin gene family can be divided into two subtypes: the membrane-associated mucins, involved in cell signaling and surface shielding, and the gel-forming mucins, responsible for the viscoelastic properties of mucus gels. Membrane-associated mucins are mostly monomeric and attach to the cell surfaces via a hydrophobic membrane-spanning domain. In contrast, gel-forming mucins possess several von Willebrand factor (vWF)-like and cysteine-rich domains that are essential for the formation of dynamic polymeric networks. Large glycans are attached to serine and threonine residues distributed throughout the apomucin. These dense O-linked oligosaccharides can contribute up to 80% of the molecular weight4. Intra- and inter-molecular disulfide bonds connecting mucin monomers ensure the integrity of the mucin gel network. As a result of heavy glycosylation and multimerization, mucins are among the largest molecules in the animal world and cannot be analyzed by standard gel electrophoresis using conventional SDS-PAGE polyacrylamide gel and standard protein ladders. These methods resolve/separate proteins with molecular weights lower than 250 kDa while mucin monomers can reach up to 2 MDa in the case of MUC16. However, high-molecular-weight protein ladders can be used to study small mucin monomers (i.e., MUC1).
A variety of techniques can be applied to study mucin size, conformation and interaction. Traditionally, biochemical characterization of mucins is accomplished by mucin isolation via isopycnic density-gradient centrifugation in denaturing buffer, followed by size-exclusion chromatography and immunodetection (e.g., slot blotting)5. Dynamic and/or multi-angle light scattering provide information on the oligomeric state of mucin-rich samples1. In addition, rate-zonal centrifugation coupled with immunodetection and transmission electron microscopy are commonly used to determine the macromolecular conformation of mucins6. Mass spectrometry is also used to quantify mucins, detect proteolytic cleavage and analyze oligosaccharide composition1,7,8. Such techniques are costly, time consuming and often require large volumes and/or high concentrations of sample. The methodology described herein, i.e., mucin separation by electrophoresis, is reproducible, low cost and can be used in high-throughput studies to provide relative mucin quantitation and investigate polymer assembly. However, this assay requires high-affinity, high-specificity mucin antibodies that may not be available for rare mucins (e.g., MUC19) or certain species (e.g., pig, ferret).
Agarose Western blotting is suitable to resolve a wide variety of mucin-rich samples with concentrations ranging from 50 &#181;g/ml (e.g., cell washes) to 5 mg/ml (e.g., sputum). This assay was introduced in the 1990s and was only performed in few specialized laboratories9,10. Initially, this technique helped identify subpopulations of mucin monomers in human respiratory secretions11,12 and confirmed the oligomerization process in goblet cells, which consists of dimer formation in the endoplasmic reticulum followed by dimer multimerization in the Golgi apparatus13. More recently, the generation of polyclonal antibodies against murine mucins facilitated studies on small animal models (e.g., mucin deficient, &#946;ENaC, OVA-challenged mouse models) and opened a new field of research for preclinical studies testing pharmacological compounds aimed at removing mucus from the lungs14-17. As a result of an increasing interest in mucin biology and the generation of novel, more specific mucin antibodies, we describe herein the methodology to separate mucins by agarose gel electrophoresis, vacuum transfer to nitrocellulose and two-color infrared fluorescent detection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>T6060-1kg</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma</td>
			<td>ARK2183-1L</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS-100g</td>
			<td>100g</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma</td>
			<td>114391-5g</td>
			<td>5g</td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>Sigma</td>
			<td>L6026-50g</td>
			<td>50g</td>
		</tr>
		<tr>
			<td>20X SSC (Sodium Saline Citrate) Buffer</td>
			<td>Promega</td>
			<td>V4261</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-1</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Trisodium Citrate (Na3C6H5O7)</td>
			<td>Sigma</td>
			<td>W302600-1kg</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>10 mM DTT (dithiothreitol)&#160;</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Milk Powder</td>
			<td>Saco</td>
			<td></td>
			<td>Instant non-fat dry milk</td>
		</tr>
		<tr>
			<td>Dulbecco Phosphate Buffered Saline (D-PBS)</td>
			<td>Gibco lifetechnologies</td>
			<td>14200-025</td>
			<td>500mL</td>
		</tr>
		<tr>
			<td>Anti-GFP Goat Primary Antibody (mouse samples)</td>
			<td>Rockland antibodies and assays</td>
			<td>600-101-215</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>UNC 222 Anti-Muc5b Rabbit Primary Antibody (mouse samples)</td>
			<td>UNC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H300 Anti-MUC5B Primary Antibodies (human samples)</td>
			<td>Santa Cruz</td>
			<td>sc-20119</td>
			<td>200ug</td>
		</tr>
		<tr>
			<td>45M1 Anti-MUC5AC Primary Antibodies (human samples)</td>
			<td>Abcam</td>
			<td>ab3649</td>
			<td>100ug</td>
		</tr>
		<tr>
			<td>Donkey Anti-rabbit 800CW IR Dye</td>
			<td>LI-COR Biosciences</td>
			<td>926-32213</td>
			<td>0.5mg</td>
		</tr>
		<tr>
			<td>Donkey Anti-mouse 680LT IR Dye</td>
			<td>LI-COR Biosciences</td>
			<td>926-68022</td>
			<td>0.5mg</td>
		</tr>
		<tr>
			<td>Electrophoresis Gel Box and Casting Tray</td>
			<td>Owl Seperation Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply Box</td>
			<td>Biorad</td>
			<td>Model 200/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Blotting Paper</td>
			<td>Amersham&#160;</td>
			<td>10600016&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Paper and Nitrocellulose membrane&#160;</td>
			<td>GE&#160;</td>
			<td>10 439 196</td>
			<td></td>
		</tr>
		<tr>
			<td>Boekel/Appligene Vacuum Blotter 230v</td>
			<td>Expotech USA</td>
			<td>230600-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Infrared Fluorescence System</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mucin agarose gel electrophoresis: western blotting for high-molecular-weight glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

We show representative results of mucin expression following agarose gel electrophoresis in BALF from the lungs of mice (Figure 1). In this example, we used the agarose gel to show upregulation of mucin production following IL-13 treatment of the Tg-Muc5ac mouse model. The Western blot shows a visual representation of mucin expression, which can be used for a quantitative analysis of multimer or monomer band signal intensity (Figure 2). This method can al...

Watch this Scientific Journal Video about Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins at JoVE.com

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

The overall goal of this western blotting technique for high-molecular-weight glycoproteins is to electrophoretically separate mucins contained in mucus-rich samples. This method can help answer key questions in the field of mucin biology, such as, investigating the polymeric state in relative concentrations of specific mucins harvested from various mucosal surfaces. The main advantage of this technique is that it is easy to establish, relatively low-cost, and requires minimal laboratory equipment.Demonstrating this procedure will be Sophia Samir, research specialist from my laboratory. Begin the experiment by preparing the gel. Pour 150 milliliters of 1x TAE 0.1%SDS buffer into a microwaveable Erlenmeyer flask to make a six-millimeter-thick gel and add 1.2 grams of agarose powder.Microwave the flask for 30-second intervals with intermittent swirling until the agarose is completely dissolved. Allow the agarose solution to cool down for up to 10 minutes. Next, place a 10 by 15 centimeter electrophoresis casting tray into a gel caster.Slowly pour the agarose solution and place the combs into position. Remove any bubbles that may have formed with a pipette tip, then allow the gel to cool and solidify. Once the gel has completely solidified, remove the comb slowly to prevent the wells from collapsing and remove the tray from the gel caster.Place the gel into the gel box and fill the electrophoresis unit until the gel is fully covered. Add 10%of loading dye to each sample. Homogenize the samples by pipetting up and down.Then, briefly spin the tubes at 5000 RPM to collect droplets. Next, use positive displacement pipettes to facilitate loading the highly viscous samples. Slowly aspirate 80 to 90 percent of the sample to avoid pipetting bubbles.Slowly load from the bottom of the well and progressively move the pipette tip upward. For samples that remain attached to the pipette tip, dispense at a 45-degree angle and elongate straight above the well or gently use the side of the well to break the stringy mucus. Accurately loading the viscoelastic samples can be challenging as mucus inherently adheres to the loading tips.Also, the buoyant force applied by the buffer on to mucus may displace total or part of the loaded volume out of the wells. Run the gel at 80 volts for 60 to 75 minutes for a double-comb gel to prevent overlap between the two rows. After the gel has run an adequate length, turn the generator power off and disconnect the electrodes from the power source.Carefully remove the gel from the gel box with one hand on either side of the casting tray to ensure the gel remains in place. Carefully place the gel flat into a glass container. Then, rinse the gel with distilled water to remove traces of TAE SDS buffer.Soak the gel with previously prepared DTT 4x SSC buffer and cover the gel. Next, gently rock the gel at 10 rotations per minute. After rocking for 20 minutes, rinse the gel with DTT-free 4x SSC buffer.Prepare the vacuum blotter for transfer by carefully cutting the nitrocellulose membrane slightly wider than the gel. Wet a porous mat with distilled water and place it smooth-side up in the blotter. Wet and place two layers of Whatman paper at the center of the porous mat.Wet a nitrocellulose membrane with DTT-free 4x SSC buffer and place it on top of the Whatman paper. Cover the porous mat with a rubber gasket leaving the opening of the gasket precisely aligned with the nitrocellulose membrane. If the porous mat is still visible, carefully adjust the membrane to ensure no gap remains between the gasket and the membrane.Wet the nitrocellulose membrane with DTT-free SSC media. Carefully place the agarose gel on top of the membrane. Ensure the gel forms a tight seal with the gasket and the wells of the gel are positioned on the membrane for transfer.Throughout protein transfer, make sure you maintain a tight seal between the agarose gel and the nitrocellulose membrane. Once the gel is placed on to the membrane, do not move or adjust the gel and keep the gel well hydrated. To minimize the formation of bubbles, squirt a few drops of 4x SSC buffer on the membrane and slide the gel from top to bottom to flush any formed bubbles.Carefully cover the gel with 4x SSC buffer. Start the mucin transfer by turning on the vacuum blotter for 90 minutes. Add a few drops of 4x SSC buffer on the gel every five to 10 minutes to prevent the gel from drying out.Upon completion, mark the wells with a pencil for orientation. Discard the gel and retrieve the nitrocellulose membrane with plastic tweezers by the edge of the membrane. Rinse the membrane immediately with 1x phosphate buffered saline or PBS.Immerse the membrane in 15 milliliters of 3%milk PBS-blocking buffer and place it on a rocker at room temperature for one hour. After incubation, discard the blocking buffer. Next, immerse the membrane in 1%milk PBS primary antibody solution and incubate the membrane overnight on a rocker at four degrees Celsius.After incubation, discard the primary antibody solution. Then, wash the membrane with PBS while rocking. Transfer the membrane to a nontransparent container and immerse the membrane in 1%milk PBS secondary antibody solution and incubate the membrane at room temperature for one hour on a rocker.After incubation, discard the secondary antibody solution. Wash the membrane three times with PBS for five minutes each. Finally, rinse the membrane with distilled water to remove salt.This western blot displays the upregulation of Muc5ac and Muc5b proteins in bronchoalveolar lavage fluids of IL-13-challenged mice compared to control mice. A quantitative analysis shows that the upregulation was 5.1 fold over PBS treated mice. Mucin agarose gel electrophoresis was used to show the reduction of mucin polymers into monomers following treatment with increasing concentrations of DTT.While attempting this procedure, it is important to remember to confirm antibody specificity before studying a particular mucin. For pharmacological testing, it is important to examine the impact that chemical reagents may have on epitope recognition. After watching this video, you should have a good understanding of how to manipulate viscoelastic samples and determine the polymeric state of mucins and their relative quantification.

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Denaturing Gradient Gel Electrophoresis DGGE

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

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 ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...