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, add 5 ml of glycerol (50%), 25 mg of bromophenol blue (0.25%), and 100 mg of sodium dodecyl sulfate (SDS) (1%).</li>
	</ol>
	</li>
	<li>Prepare 2 liters of 20x sodium saline citrate (SSC) buffer.
	<ol>
		<li>In 1.5 liters of dH2O, add 350.6 g of NaCl (3 M) and 176.4 g of trisodium citrate (Na3C6H5O7) (0.3 M). Adjust pH to 7.0 and make up the volume to 2 liters with dH2O.</li>
	</ol>
	</li>
	<li>Prepare 1 liter of 1x TAE-0.1% SDS buffer.
	<ol>
		<li>Add 20 ml of 50x TAE to 980 ml of dH2O. Add 1 g of SDS to make a 0.1% SDS-TAE solution.</li>
	</ol>
	</li>
</ol>
2. TAE-SDS Agarose Gel Preparation
<ol>
	<li>Pour sufficient volume (i.e., 150 ml) of 1x TAE-0.1% SDS buffer in a microwavable Erlenmeyer flask to make a 5 - 7 mm-thick gel. Add 0.8% (1.2 g) agarose powder.</li>
	<li>Microwave flask in 30 sec intervals with intermittent swirling until agarose is completely dissolved (usually 1 - 3 min). Use hot hand pads during this process. Be careful of eruptive boiling while swirling.</li>
	<li>Allow the agarose solution to cool down for 5 - 10 min. Note: The agarose solution will be ready to be poured when bottom of the flask can be left on the palm for 5 sec.</li>
	<li>Prepare electrophoresis casting tray (10 x 15 cm) by sealing the edges with tape and placing well comb in position.</li>
	<li>Pour agarose solution slowly in the casting tray to avoid the formation of bubbles. Remove bubbles using a pipette tip. Wait for the gel to cool and completely solidify. Once solidified, remove the comb slowly to avoid collapsing the wells by suction and remove the tape from the edges of the tray.</li>
	<li>Place agarose gel into the gel box and fill the electrophoresis unit with 1x TAE-0.1% SDS buffer until the gel is fully covered.</li>
</ol>
3. Sample Loading and Electrophoresis Separation
Note: In our laboratory, we commonly use samples from both mouse and humans, including bronchoalveolar lavage fluid (BALF; both species), cell washings from human bronchial epithelial cells (HBE), and human saliva and sputum samples. Samples can be denatured in 6 M urea upon collection or stored for short periods of time (6 hr) by adding proteinase cocktail inhibitor.&#160;
<ol>
	<li>Prepare samples for loading by adding 10% of loading dye to each sample (i.e., 30 &#181;l of sample and 3 &#181;l of dye). Homogenize samples by pipetting up and down. Briefly spin down tubes at 5,000 rpm to collect droplets.</li>
	<li>Carefully load equal volume of samples into the wells.
	<ol>
		<li>Pre-wet tips and/or use positive displacement pipettes to facilitate loading of highly viscous samples. Slowly aspirate 80 - 90% of sample-dye solution (i.e., 27 - 30 &#181;l) to avoid pipetting bubbles.</li>
		<li>Slowly load at the bottom of the wells and progressively move the pipet tip upward. For samples that remain attached to the pipette tip, dispense at a 45&#176; angle and elongate straight above the well or gently use the side of the well to break the stringy mucus. Do not overload the wells.</li>
	</ol>
	</li>
	<li>Connect the electrodes from the gel box to the electrophoresis power generator. Ensure the electrodes are connected properly (i.e., red lead plugged to the positive output of the generator) to allow negatively charged mucins to run towards the positive electrode.</li>
	<li>Run the gel at 80 Volts for 90 min for a single comb gel, or 60 - 75 min for a double comb gel to prevent overlap between the two rows.</li>
	<li>Turn generator power OFF and disconnect the electrodes from the power source.</li>
	<li>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.</li>
</ol>
4. Reduce Agarose Gel for Efficient Mucin Transfer
<ol>
	<li>Carefully place the gel flat in a glass container. Rinse the gel with dH2O to remove traces of TAE-SDS buffer.</li>
	<li>Prepare 1 liter of 4x SSC buffer by adding 200 ml of 20x SSC into 800 ml of dH2O. Make 200 ml of 10 mM dithiothreitol (DTT) 4x SSC solution by adding 309 mg of fresh DTT into 200 ml of 4x SSC buffer.</li>
	<li>Soak the gel with DTT-4x SSC buffer and cover. Gently rock for 20 min (10 rotations per minute). Rinse gel with DTT-free 4x SSC buffer.</li>
</ol>
5. Vacuum Blotter Assembly and Sample Transfer to a Nitrocellulose Membrane
<ol>
	<li>Prepare vacuum blotter for transfer.
	<ol>
		<li>Carefully cut nitrocellulose membrane at dimensions slightly wider than the gel (i.e., 10 x 15 cm).</li>
		<li>Wet porous mat with dH2O and place it smooth-side-up in the blotter.</li>
		<li>Wet nitrocellulose membrane with DTT-free 4x SSC buffer and place it at the center of the porous mat.</li>
		<li>Cover the porous mat with the rubber gasket, leaving the opening of the gasket precisely aligned with the nitrocellulose membrane. If porous mat is still visible, carefully adjust the membrane to ensure no gap remains between gasket and membrane.</li>
	</ol>
	</li>
	<li>Carefully place the agarose gel on top of the membrane. Ensure gel forms a tight seal with the gasket and the wells of the gel are positioned on the membrane for transfer.
	<ol>
		<li>Avoid the formation of bubbles between the membrane and the gel. To minimize the formation of bubbles, squirt few drops of 4x SSC buffer on the membrane and slide the gel from top to bottom to flush any formed bubbles. Avoid moving the gel on the membrane as proteins will begin to blot immediately.</li>
	</ol>
	</li>
	<li>Carefully cover the gel with 4x SSC buffer.</li>
	<li>Begin mucin transfer by suction.
	<ol>
		<li>Turn vacuum blotter ON and set pressure at 40 - 50 mBar. Add a few drops of 4x SSC buffer on the gel every 5 - 10 min to prevent the gel from drying out. Run transfer for 90 min. Optional: Upon completion, mark the wells with a pencil for orientation.</li>
	</ol>
	</li>
	<li>Dispose of the gel and retrieve nitrocellulose membrane using plastic tweezers on the edge of the membrane.</li>
</ol>
6. Membrane Blocking and Detection
<ol>
	<li>Rinse membrane immediately with 1x phosphate-buffered saline (PBS). Do not let the membrane dry out.</li>
	<li>Immerse the membrane into 50 ml of 3% milk-PBS blocking buffer and place on a rocker at room temperature for 1 hr. After incubation, discard the blocking buffer.</li>
	<li>Immerse membrane in 1% milk-PBS primary antibody solution. Incubate the membrane in the primary antibody solution overnight on a rocker at 4 &#176;C. After incubation, discard the primary antibody solution. Dilute the primary antibodies to a 1:2,000 ratio of antibody solution to blocking buffer. Note: For samples originating from mice, we use UNC 222 rabbit anti-Muc5b and goat anti-Green Fluorescent Protein (GFP). For samples originating from humans, we use H300 anti-MUC5B and 45M1 anti-MUC5AC primary antibodies.</li>
	<li>Wash membrane 3 times with PBS for 10 min on a rocker.</li>
	<li>Immerse membrane in 1% milk-PBS secondary antibody solution. Dilute the secondary antibodies to a 1:7,500 ratio of antibody solution to blocking buffer (e.g., 700 anti-rabbit and 680 anti-goat) and incubate at room temperature for 1 hr on a rocker. Protect from light by incubating in a non-transparent container. Discard the secondary antibody solution after 1 hr.</li>
	<li>Wash membrane 3 times with PBS for 5 min. Rinse membrane with dH2O to remove salt.</li>
	<li>Read the membrane on an infrared fluorescence system according to manufacturer&#39;s instructions.</li>
</ol>

<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 authors have no conflict to disclose.

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><tbody><tr><td>Tris Base</td><td>Sigma</td><td>T6060-1kg</td><td>1 kg</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma</td><td>ARK2183-1L</td><td>1 L</td></tr><tr><td>EDTA</td><td>Sigma</td><td>EDS-100g</td><td>100 g</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-1</td><td>1 L</td></tr><tr><td>Bromophenol Blue</td><td>Sigma</td><td>114391-5g</td><td>5 g</td></tr><tr><td>SDS (Sodium Dodecyl Sulfate)</td><td>Sigma</td><td>L6026-50g</td><td>50 g</td></tr><tr><td>20x SSC (Sodium Saline Citrate) Buffer</td><td>Promega</td><td>V4261</td><td>1 L</td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271-1</td><td>1 kg</td></tr><tr><td>Trisodium Citrate (Na&lt;sub&gt;3&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</td><td>Sigma</td><td>W302600-1kg</td><td>1 kg</td></tr><tr><td>10 mM DTT (dithiothreitol)&amp;nbsp;</td><td>Sigma</td><td>D0632</td><td>25 g</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>500 ml</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>200 &amp;#956;g</td></tr><tr><td>45M1 Anti-MUC5AC Primary Antibodies (human samples)</td><td>Abcam</td><td>ab3649</td><td>100 &amp;#956;g</td></tr><tr><td>Donkey Anti-rabbit 800CW IR Dye</td><td>LI-COR Biosciences</td><td>926-32213</td><td>0.5 mg</td></tr><tr><td>Donkey Anti-mouse 680LT IR Dye</td><td>LI-COR Biosciences</td><td>926-68022</td><td>0.5 mg</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&amp;nbsp;</td><td>10600016&amp;nbsp;</td><td></td></tr><tr><td>Whatman Paper and Nitrocellulose membrane&amp;nbsp;</td><td>GE&amp;nbsp;</td><td>10 439 196</td><td></td></tr><tr><td>Boekel/Appligene Vacuum Blotter 230 v</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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25.4K Views.  University of North Carolina at Chapel Hill. 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. 

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

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Biochemistry

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 ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

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 ...

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 ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

Bioengineering

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

Two-Dimensional Gel Electrophoresis

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

Education

JoVE Science Education

Chemistry

Polyacrylamide Gel Electrophoresis: An Analytical Technique to Detect Heparan Sulfates of Varying Chain Lengths Isolated from Mouse Lung Tissue

Source: LaRiviere, W. B. et al., <a href="https://www.jove.com/v/62319">Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining</a>. J. Vis. Exp. (2021)
This video demonstrates the separation procedure of heparan sulfates isolated from mouse lung tissue using polyacrylamide gel electrophoresis.

Source: LaRiviere, W. B. et al., Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining. J. Vis. Exp. (2021)
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

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

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

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

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

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