JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: June 14th, 2016

DOI:

10.3791/54153

1Marsico Lung Institute/CF Center, University of North Carolina at Chapel Hill, 2Telethon Kids Institute, University of Western Australia, 3Department of Pediatrics, 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.

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.

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,....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Prepare Buffers for Mucin Gel Western Blotting

  1. Prepare 1 liter of 50x TAE (Tris-acetate-EDTA) buffer.
    1. 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).
    2. Adjust pH to 8.0 and make the volume up to 1 liter with dH2O.
  2. Prepare 10 ml of 10x loading buffer.
    1. Prepare 5 ml of 1x TAE buffer. To do this, ad.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

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

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved