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Protocol

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Biology

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Published: February 25th, 2021

DOI:

10.3791/62319

1Department of Medicine, University of Colorado Anschutz Medical Campus, 2Medical Scientist Training Program, University of Colorado Anschutz Medical Campus, 3Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 4Department of Health Sciences, Curtin University, 5Department of Medicine, Denver Health Medical Center

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 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.

Glycosaminoglycans (GAGs) are a diverse family of linear polysaccharides that are a ubiquitous element in living organisms and contribute to many basic physiological processes1. GAGs such as heparan sulfate (HS) and chondroitin sulfate (CS) may be sulfated at distinct positions along the polysaccharide chain, imparting geographic domains of negative charge. These GAGs, when tethered to cell-surface proteins known as proteoglycans, project into the extracellular space and bind to cognate ligands, allowing for the regulation of both cis- (ligand attached to the same cell) and trans- (ligand attached to neighboring cell) signaling processes

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All biological samples analyzed in this protocol were obtained from mice, under protocols approved by the University of Colorado Institutional Animal Care and Use Committee.

1. Heparan sulfate isolation

  1. Delipidation of tissue samples
    NOTE: Delipidation is an optional step for fat-rich tissues.
    1. Make a 1:1 mixture of methanol and dichloromethane. Prepare approximately 500 μL per sample; larger pieces of tissue may require up to 1 mL.
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Alcian blue is used to stain sulfated GAGs 10; this signal is amplified by use of a subsequent silver stain 11. Figure 1 provides a visual demonstration of the silver staining development process. As demonstrated, the Alcian blue signal representing GAGs separated by electrophoresis is amplified as the developing agent penetrates the polyacrylamide gel. Typically, the developing process will reduce silver and Alcian blue-stained GAGs in a densi.......

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GAGs play a central role in many diverse biological processes. One of the principal functions of sulfated GAGs (such as HS and CS) is to interact with and bind to ligands, which can alter downstream signaling functions. An important determinant of GAG binding affinity to cognate ligands is the length of the GAG polymer chain 8,9,14. For this reason, it is important for researchers to be able to define with reasonable precision t.......

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This work was funded by F31 HL143873-01 (WBL), R01 HL125371 (RJL and EPS)

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Name Company Catalog Number Comments
Accuspin Micro17 benchtop microcentrifuge thermoFisher Scientific 13-100-675 Any benchtop microcentrifuge/rotor combination capable of 14000 xG is appropriate
Acrylamide (solid) thermoFisher Scientific BP170-100 Electrophoresis grade
Actinase E Sigma Aldrich P5147 Protease mix from S. griseus
Alcian Blue 8GX (solid) thermoFisher Scientific AC400460100
Ammonium acetate (solid) thermoFisher Scientific A639-500 Molecular biology grade
Ammonium hydroxide (liquid) thermoFisher Scientific A669S-500 certified ACS
Ammonium persulfate (solid) thermoFisher Scientific BP179-25 electrophoresis grade
Barnstead GenPure Pro Water Purification System ThermoFisher Scientific 10-451-217PKG Any water deionizing/ purification system is an acceptable substitute
Boric acid (solid) thermoFisher Scientific A73-500 Molecular biology grade
Bromphenol blue (solid) thermoFisher Scientific B392-5
Calcium acetate (solid) ThermoFisher Scientific 18-609-432 Molecular biology grade
Calcium chloride (solid) ThermoFisher Scientific AC349610250 Molecular biology grade
CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) ThermoFisher Scientific 28299
Chondroitinase ABC Sigma Aldrich C3667
Criterion empty cassette for PAGE (1.0mm thick, 12+2 wells) Bio-Rad 3459901 Any 1.0mm thick PAGE casting cassette system will suffice
Criterion PAGE Cell system (cell and power supply) Bio-Rad 1656019 any comparable vertical gel PAGE system will work)
Dichloromethane (liquid) thermoFisher Scientific AC610931000 certified ACS
EDTA disodium salt (solid) thermoFisher Scientific 02-002-786 Molecular biology grade
Glacial acetic acid (liquid) thermoFisher Scientific A35-500 Certified ACS
Glycine (solid) thermoFisher Scientific G48-500 Electrophoresis grade
Heparanase I/III Sigma Aldrich H3917 From Flavobacterium heparinum
Heparin derived decasaccharide (dp10) galen scientific HO10
Heparin derived hexasaccharde (dp6) Galen scientific HO06
Heparin derived oligosaccharide (dp20) galen scientific HO20
Hydrochloric acid (liquid) thermoFisher Scientific A466-250
Lyophilizer Labconco 7752020 Any lyophilizer that can achieve -40C and 0.135 Torr will work; can also be replaced with rotational vacuum concentrator
Methanol (liquid) thermoFisher Scientific A412-500 Certified ACS
Molecular Imager Gel Doc XR System Bio-Rad 170-8170 Any comparable gel imaging system is an acceptable substitute
N,N'-methylene-bis-acrylamide (solid) thermoFisher Scientific BP171-25 Electrophoresis grade
Phenol red (solid) thermoFisher Scientific P74-10 Free acid
Q Mini H Ion Exchange Column Vivapure VS-IX01QH24 Ion exchange column must have minimum loading volume of 0.4mL, working pH of 2-12, and selectivity for ionic groups with pKa of 11
Silver nitrate (solid) thermoFisher Scientific S181-25 certified ACS
Sodium Acetate (solid) ThermoFisher Scientific S210-500 Molecular biology grade
Sodium chloride (solid) thermoFisher Scientific S271-500 Molecular biology grade
Sodium hydroxide (solid) thermoFisher Scientific S392-212
Sucrose (solid) thermoFisher Scientific BP220-1 Molecular biology grade
TEMED (N,N,N',N'-tetramethylenediamine) thermoFisher Scientific BP150-20 Electrophoresis grade
Tris base (solid) thermoFisher Scientific BP152-500 Molecular biology grade
Ultra Centrifugal filters, 0.5mL, 3000 Da molecular weight cutoff Amicon UFC500324 Larger volume filter units may be used, depending on sample size. 
Urea (solid) ThermoFisher Scientific 29700
Vacufuge Plus Eppendorf 22820001 Any rotational vacuum concentrator will work; can be replaced with lyophilizer
Vacuum filter unit, single use, 0.22uM pore PES, 500mL volume thermoFisher Scientific 569-0020 Alternative volumes and filter materials acceptable

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