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 processes2. Furthermore, GAGs also perform critical roles as structural elements in tissues such as the glomerular basement membrane3, the vascular endothelial glycocalyx4 and pulmonary epithelial glycocalyx5, and in connective tissues such cartilage6.
The length of GAG polysaccharide chains varies substantially according to its biological context and can be dynamically lengthened, cleaved, and modified by a highly complex enzymatic regulatory system7. Importantly, the length of GAG polymer chains contributes substantially to their binding affinity for ligands and, subsequently, to their biological function8,9. For this reason, determination of the function of an endogenous GAG requires appreciation of its size. Unfortunately, unlike proteins and nucleic acids, very few readily available techniques exist to detect and measure GAGs, which has historically resulted in relatively limited investigation into the biological roles of this diverse polysaccharide family.
This article describes how to isolate and purify GAGs from most biological tissues, and, also, describes how to use polyacrylamide gel electrophoresis (PAGE) to evaluate the length of the isolated polymers with a fair degree of specificity. In contrast to other, highly complex (and often mass spectrometry-based) glycomic approaches, this method can be employed using standard laboratory equipment and techniques. This practical approach may, therefore, expand investigators' ability to determine the biological role of native GAGs and their interaction with contextually important ligands.
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
2. Polyacrylamide gel electrophoresis of isolated and purified glycosaminoglycans
3. Silver staining protocol
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 density dependent fashion, with the edges of each band reducing first while the more densely staining regions in the center will stain last.
In the literature, the reported limit of detection of GAGs using PAGE-based approaches range from 0.5-1 µg12,13. To determine the limit of detection using our approach, heparan sulfate oligosaccharides of different polymer lengths (unfractionated heparin, dp20, dp10, dp6) were loaded onto a 22% polyacrylamide gel, then ran and stained as described above. Each oligosaccharide was loaded twice at two different masses: 1.0 µg and 0.5 µg. Figure 2 demonstrates that our technique can quite readily detect 0.5 µg of purified GAG. Notably, unfractionated heparin was least readily detected of the four different oligosaccharides tested, likely due to a wider distribution of polymer sizes that correspondingly reduces the density of each individual band.
To assess the efficiency of GAG purification from liquid biological samples, GAGs were isolated from two bronchoalveolar lavage (BAL) samples. As shown in Figure 3, the first sample marked as A used for GAG isolation was 1 mL of BAL fluid harvested from a mouse 24 h after intratracheal lipopolysaccharide (LPS) (3 mg/kg), administered to induce alveolar epithelial HS shedding 5. 10 µg of commercially available dp6 was added directly to this BAL fluid to serve as a "spike in" control to assess loss of GAGs during the isolation process. The second sample marked as B consisted of 10 mL of BAL fluid pooled from 3 mice who were given intratracheal LPS (3 mg/kg) 24 h prior, without exogenous GAG spike-in. Both samples were processed for GAG isolation simultaneously, and all 3 fractions eluted from the ion exchange column were retained and further processed in order to determine if any GAGs were present in the low-affinity and wash fractions. 2 µg of commercially purchased dp20, dp10, and dp6 heparan sulfate oligosaccharides were run in the PAGE gel alongside these samples to provide a reference by which to qualitatively assess the size of HS GAGs in each eluted fraction, as well as a reference to compare density with the 10 µg "spike in" control that underwent GAG isolation.
To demonstrate the use of this technique on solid tissue, heparan sulfate was isolated from a 15 mg piece of frozen mouse lung as described above. During the isolation and purification process, the low affinity fraction (0.2 M NaCl) eluted off the ion exchange column was retained and processed alongside the high affinity (2.7 M NaCl) fraction and run on the gel (Figure 4). 2 µg of commercially purchased dp20, dp10, and dp6 heparan sulfate oligosaccharides were run alongside these samples to provide a reference for size. As can be seen, the whole lung homogenate yielded an ample quantity of isolated HS, with the smallest fragments approximately equaling dp10 in size. The relative enrichment of Alcian blue/silver stain avid content GAGs in the 2.7 M NaCl fraction demonstrates that heparan sulfate binds with high affinity to the ion exchange columns and can be eluted off the column with high specificity. The whole lung homogenate yielded an ample quantity of isolated heparan sulfate (2.7 M NaCl fraction), with the smallest fragments approximately equaling dp10 in size.
Figure 1: Silver stain development process. Alcian blue staining of unfractionated heparin (UFH) or size-defined oligosaccharides (dp = degree of polymerization) separated by electrophoresis is amplified by silver staining. From left to right: UFH, dp20, dp10, dp6. Please click here to view a larger version of this figure.
Figure 2: Sensitivity of polyacrylamide gel and silver stain for the detection for heparan sulfate. Heparan sulfate oligosaccharides of different lengths (unfractionated heparin aka UFH, dp20, dp10, dp6) were onto a 22% polyacrylamide gel and ran and silver stained. Each oligosaccharide was loaded twice at two different masses: 1.0 µg (leftmost bands) and 0.5 µg (rightmost bands). Please click here to view a larger version of this figure.
Figure 3: Efficiency of GAG purification from liquid biological samples. GAGs isolated from two bronchoalveolar lavage (BAL) samples, labeled here as "A" and "B", were run on a 22% polyacrylamide gel and silver stained. Sample A consisted of 1mL of BAL fluid harvested from a mouse 24 h after treatment with 3 mg/kg intratracheal lipopolysaccharide (LPS). An additional 10 µg of dp6 HS were added to this sample as a "spike in" control. Sample B consisted of 10 mL of pooled BAL fluid collected from 3 mice 24 h after administration of LPS. Each sample was run alongside fractions from the ion exchange column eluted with either diluent ("wash") or low concentrations of NaCl (0.2 M). 2 µg of commercially purchased dp20, dp10, and dp6 heparan sulfate oligosaccharides were used as size references (leftmost bands). Please click here to view a larger version of this figure.
Figure 4: Measuring the size of heparan sulfates isolated and purified from a healthy mouse lung. Heparin sulfate content isolated and purified from 15 mg frozen sample of a lung isolated from a healthy mouse and run on a 22% polyacrylamide gel. Heparan sulfate was eluted from the ion exchange column with either low concentrations (0.2 M; low affinity fraction) or high concentrations (2.7 M, 16% solution; high affinity fraction) of NaCl. 2 µg of commercially purchased dp20, dp10, and dp6 heparan sulfate oligosaccharides were used as size references (leftmost bands). Please click here to view a larger version of this figure.
NOTE: All solutions must be filtered (0.22μm) before use. | |||
Solution | Recipe | Composition | Comments |
Resolving gel/lower chamber running buffer (2 L or 4 L) | Boric acid, MW 61.83 | 2L: 12.36 g; 4L: 24.76 g | Desired concentration: 0.1 M |
Tris base, MW 124.14 | 2L: 24.2 g; 4L: | Desired concentration: 0.1 M | |
Disodium EDTA MW 336.21 or dihydrate, MW 372.36 | 2L: 6.7 g or 7.4 g, respectively; 4L 13.4 g or 14.8 g, respectively | Desired concentration: 0.01 M | |
Deionized water | 2L or 4L | Adjust pH to 8.3 after fully dissolving reagents | |
Upper chamber running buffer (1 L) | Glycine | 93 g | Desired concentration: 1 M |
Tris base, MW 124.14 | 24.2 g | Desired concentration: 0.2 M | |
Deionized water | 1 L | ||
Resolving gel, 22% total acrylamide (500mL) | Acrylamide, MW 71.08 | 100.1 g | Desired concentration: 20.02% w/v |
N,N'-methylene-bis-acrylamide (bis, MW 154.17) | 10 g | Desired concentration: 2% w/v | |
Sucrose | 75 g | Desired concentration: 15% w/v | |
Resolving gel buffer | 500 mL | Bring to a total volume of 500mL; will require less than 500mL of buffer total | |
Resolving gel, 15% total acrylamide (400mL) | Acrylamide, MW 71.08 | 56.3 g | Desired concentration: 14.08% w/v |
N,N'-methylene-bis-acrylamide (bis, MW 154.17) | 3.7 g | Desired concentration: 2% w/v | |
Sucrose | 20.8 g | Desired concentration: 15% w/v | |
Resolving gel buffer | 400 mL | Bring to a total volume of 400mL; will require less than 400mL of buffer total | |
Stacking gel (100 mL) | Acrylamide, MW 71.08 | 4.75 g | Desired concentration: 4.75% w/v |
N,N'-methylene-bis-acrylamide (bis, MW 154.17) | 0.25 g | Desired concentration: 0.25% w/v | |
Resolving gel buffer | 100 mL | Add 80mL buffer and full dissolve reagents. Then adjust pH to 76.3 with hydrochloric acid, dropwise. Then bring to a total volume of 100 mL with resolving gel buffer. | |
Sample loading buffer (500 mL) | Sucrose | 250g | Desired concentration: 50% w/v |
Phenol red | 500mg | Desired concentration: 1mg/mL | |
Bromophenol blue | 250mg | Desired concentration: 0.5mg/mL | |
Deionized water | 500mL | Bring to a total volume of 500mL; will require less than 500mL of water total | |
Heparin derived oligosacharide 'ladder' (2mL each) | dp6 | 0.1mg | NOTE: Recommend using heparin derived oligosaccharides 6 polymer subunits in length (aka degree of polymerization 6, or dp6) for the smallest band, dp20 for the largest band, and dp10 for middle band. However, other combinations can be used if desired. Desired concentration: 0.05 mg/mL |
dp10 | 0.1mg | ||
dp20 | 0.1mg | ||
Deionized water | 3mL | Dissolve each oligosaccharide in separate tubes with 1mL each | |
Sample loading buffer | 3mL | Add 1mL (1:1 mixture) to each oligosaccharide solution |
Table 1: Solutions required for polyacrylamide gel electrophoresis of purified glycosaminoglycans. All solutions must be filtered (0.22 µm) before use.
NOTE: All solutions must be filtered (0.22μm) before use. | |||
Solution | Recipe | Composition | Comments |
Alcian blue staining solution (500mL) | Alcian blue 8GX powder | 2.5g | Desired concentration: 0.5% w/v |
2% v/v glacial acetic acid | 500mL | ||
Silver stain (200 mL) | Deionized water | 192.7 mL | Prepare stain solution fresh; use within one week. |
7.6M sodium hydroxide | 2 mL | To make, add 3.04 g sodium hydroxide pellets to 10 mL water | |
Ammonium hydroxide | 3.3 mL | ||
4M silver nitrate solution | 2 mL | To make, add 3.397 g silver nitrate to 5 mL water. Add dropwise while stirring to avoid precipitation. | |
Developing solution (501 mL) | Deionized water | 500 mL | Developing solution must be made fresh and used within 24 h. |
2.5% w/v citric acid | 1 mL | To make, add 100mg to 4 mL deionized water. Citric acid must be made fresh. | |
Formaldehyde | 250μL | ||
Stop solution (500 mL) | Deionized water | 300 mL | |
Glacial acetic acid | 20 mL | ||
Methanol | 90 mL |
Table 2: Solutions required for silver staining of glycosaminoglycans separated by polyacrlamide gel electrophoresis. All solutions must be filtered (0.22 µm) before use.
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 the size of GAG chains isolated from biological samples of interest. To be practical, this technique should be capable of being performed using common laboratory equipment and reagents.
This protocol describes a method to isolate and purify GAGs from biological samples, to separate them by size via PAGE, and to visualize them using Alcian blue and silver staining technique. While there are several ways to separate glycosaminoglycans by size, our approach has several strengths specific to the application of this technique in life science laboratories. Firstly, with a limit of detection of 0.5 µg, this technique is highly sensitive in the detection of GAGs of interest. In our experience, even samples that contain relatively low concentrations of GAGs (i.e., BAL fluid samples) should yield more than enough GAG for detection using this method. While the yield from isolation and purification of BAL fluid will vary by technique and specific GAG of interest, it has been our experience that 10 mL of raw BAL fluid yields sufficient HS to be detectable using this technique. The yield from solid organs is substantially higher, depending on the tissue harvested, but it is our experience that an initial solid sample of 10 mg will yield ample HS for detection using this technique.
It is important to note that the final imaging of the stained PAGE gel will vary according to the imaging technologies available to the end-user. Digital photographs of the gel can be taken using a number of different systems, including numerous commercially available gel documentation systems or a regular commercial camera, depending the available equipment and the sensitivity of detection required (typically dictated by the amount of sample loaded onto the gel). It also should be noted that the light source required to digitally image this gel may vary depending on the density of the sample and the amount of development time required during the staining process. Gels that develop rapidly and produce silver-stained bands readily visible to the naked eye will require UV transillumination for the best images, as the majority of the light will be absorbed by unreacted Alcian blue. Gels that require longer development times (such as those with very small amounts of sample) will require either epi-illumination or transillumination with normal full spectrum light, as this will be best absorbed by the silver stain.
A further strength of this technique is that it is particularly adaptable to life sciences labs, due to its basis in simple PAGE technology, using equipment and reagents that are commonly available and cheaply acquired. While there are other approaches to quantifying the length of isolated GAG polymers (e.g., capillary electrophoresis), they typically require both knowledge and equipment that is not commonly available in most life sciences laboratories15,16. The simplicity of this approach and the relatively affordable and available nature of the reagents required makes this technique readily adaptable by life sciences researchers interested in studying GAG biology in the context of their given subfields. Furthermore, this technique serves as an essential complement to mass spectrometry-based techniques of detecting GAGs in biological tissues. While mass spectrometry based approaches are able to detect GAGs with high sensitivity and to discern subtle differences in structure from complex samples17, due to the nature of the technology it is not able to distinguish between GAG polymers by size. For this reason, a PAGE based approach is essential to divining the size of HS polymers in biological samples of interest.
It is important to note that there are several limitations to the techniques described in this paper. The first and most salient is that because of the charge-charge interaction that are central to the Alcian blue staining reaction, this approach is highly selective for highly sulfated GAGs and will only weakly stain more neutrally-charged moieties (e.g., hyaluronic acid18). Thus, this technique is likely to bias its results towards more acidic GAG moieties. For this reason, complementary approaches to measure GAG content in biological samples of interest (e.g., mass spectrometry-based techniques) should be used adjunctively to provide a more complete picture of the GAGs present in experimental samples. Furthermore, for end-users specifically interested in measuring the size of hyaluronan, others have described similar PAGE based techniques that leverage biotinylated hyaluronic acid binding protein (HABP) which may be adaptable to the basic technique described here19.
In summary, the technique presented in this article can be used to isolate, purify, and detect GAGs from biological samples with a great deal of sensitivity and specificity as well as to measure the native length of these polysaccharide chains. This information can be critical to testing hypotheses about GAG-ligand interactions due to the importance of GAG polymer length in determining cognate ligand binding affinity. This approach has several advantages, most notably its relative simplicity and adaptability to life sciences research laboratories, though it is limited by its relative bias towards negatively charged GAG moieties. Despite this drawback, this technique represents a robust tool that would encourage investigators to study the role of GAGs in organ homeostasis and disease.
The authors have nothing to disclose.
This work was funded by F31 HL143873-01 (WBL), R01 HL125371 (RJL and EPS)
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 |