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

Summary

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.

Abstract

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.

Introduction

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,....

Protocol

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, add 5 ml of glycerol (50%), 25 mg of bromophenol blue (0.25%), and 100 mg of sodium dodecyl sulfate (SDS) (1%).
3. Prepare 2 liters....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tris Base	Sigma	T6060-1kg	1 kg
Glacial Acetic Acid	Sigma	ARK2183-1L	1 L
EDTA	Sigma	EDS-100g	100 g
Glycerol	Fisher	BP229-1	1 L
Bromophenol Blue	Sigma	114391-5g	5 g
SDS (Sodium Dodecyl Sulfate)	Sigma	L6026-50g	50 g
20x SSC (Sodium Saline Citrate) Buffer	Promega	V4261	1 L
NaCl	Fisher	S271-1	1 kg
Trisodium Citrate (Na3C6H5O7)	Sigma	W302600-1kg	1 kg
10 mM DTT (dithiothreitol)	Sigma	D0632	25 g
Milk Powder	Saco		Instant non-fat dry milk
Dulbecco Phosphate Buffered Saline (D-PBS)	Gibco lifetechnologies	14200-025	500 ml
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	200 μg
45M1 Anti-MUC5AC Primary Antibodies (human samples)	Abcam	ab3649	100 μg
Donkey Anti-rabbit 800CW IR Dye	LI-COR Biosciences	926-32213	0.5 mg
Donkey Anti-mouse 680LT IR Dye	LI-COR Biosciences	926-68022	0.5 mg
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 230 v	Expotech USA	230600-2
Odyssey Infrared Fluorescence System	LI-COR Biosciences