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 ...

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