Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 function^8,9. 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.

Protokół

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.
   2. Place each tissue sample into a small glass container with a lid for delipidation.
      NOTE: Sample mass may vary per tissue of interest and experimental needs. 50 mg or less is typically sufficient for adequate GAG yield, but some optimization may be required by the end-user. 
   3. Add 500 μL of the methanol:dichloromethane solution to each glass container and mix. Ensure all solid tissue samples are completely submerged in solvent.
      NOTE: Use serological pipettes or plastic conical tubes to mix and handle the methanol/dichloromethane solution; other plastics may dissolve.
   4. Place samples on a shaker (in secured rack) in a chemical fume hood and agitate gently for 1 h.
   5.  Agitate the samples to mix and centrifuge at 17,000 x g for 5 min at 4 °C.
   6. Carefully pipette out the organic solvent fraction (supernatant). This is the lipid fraction - it does not contain GAGs and can be discarded.
      NOTE: Try not to disturb the tissue as small pieces could be lost. It is preferable to leave some of the organic layer in the sample container, rather than risking the sample loss.
   7. Leave the lid of the glass container open and let it evaporate overnight in the hood. Change the tube for the next step as those tubes will not survive further processing.
   8. Once the solvent mixture has fully evaporated, proceed to mechanical disintegration (if residual sample is >50 mg) or Actinase E digestion (< 50 mg). 
2. Mechanical disintegration of solid tissue (optional; for larger tissue samples)
   NOTE: Most smaller pieces of solid tissue (approximately 50 mg or less) should dissolve completely during the digestion step. However, larger samples will require mechanical disintegration.
   1. Flash-freeze samples of interest by placing them in an appropriately sized polypropylene tube and placing the closed tube into liquid nitrogen. Allow the sample to freeze until completely solid.
   2. Using a clean mortar and pestle, grind frozen samples into a powder-like consistency.
   3. Proceed directly to Actinase E digestion (Step 1.4).
3. Sample desalting and concentration
   NOTE: This is an optional step and only required for dilute liquid tissue samples (e.g., broncho-alveolar lavage fluid (BALF) or plasma).
   1. Pool liquid samples into appropriate experimental groups (e.g., by biological replicate, or experimental group)
   2. Place total sample volume into a 500 μL centrifugal filter column with a molecular weight cut-off (MWCO) of 3,000 Da.
   3. Spin for 30 min at 14,000 x g at room temperature. Repeat as needed if the desired sample volume exceeds capacity of the centrifugal filter column.
   4. Wash each column 3x with 400 μL of deionized, filtered water. Discard the flow through.
   5. Invert the filter and spin for 1 min at 2,000 x g in fresh, appropriately labeled collection tubes. Freeze at -80 °C or proceed to next step.
4. Actinase E digestion
   NOTE: This step is required to digest and ultimately remove protein contaminants from your sample.
   1. Mix samples 1:1 with recombinant Actinase E to a desired concentration of 10 mg/mL. Add appropriate volume of 10x digestion buffer concentrate. Desired final concentration: 0.005 M calcium acetate and 0.01 M sodium acetate, pH 7.5. For example: 190 μL of liquid sample, 190 μL of 20 mg/mL Actinase E, 20 μL of 0.05 M calcium acetate, 0.1 M sodium acetate.
   2. Agitate samples gently to mix, then digest for 48-72 h at 55 °C (up to 7 days for whole tissue).
   3. Heat to 80 °C for 15-20 min to heat inactivate Actinase E. 
   4. Freeze the sample at -80 °C or continue to step 1.5.
5. Sample desalting and concentration
   NOTE: If a low mass of the GAG of interest is anticipated in the biological sample, or if conservation of consumable reagents (i.e., centrifugal filter columns) is desirable, samples can be pooled to produce a single concentrate per experimental group (e.g., by biological replicate, or experimental group).
   1. Place total sample volume into a 500 μL centrifugal filter column with a MWCO of 3,000 Da.
   2. Spin for 30 min at 14,000 x g at room temperature. Repeat as needed, if the desired sample volume exceeds capacity of the centrifugal filter column.
   3. Wash each column 3x with 400 μL deionized, filtered water. Discard the flow through.
   4. Invert filter and spin for 1 min at 2,000 x g in fresh, appropriately labeled collection tubes (generally included with the centrifugal filter columns). Freeze at -80 °C or proceed to next step.
6. Desiccation
   1. Either place the dissolved samples from step 1.5.5 in a rotational vacuum concentrator overnight or lyophilize the samples as detailed below.
   2. Freeze samples thoroughly either overnight in -80 °C or by dipping in liquid nitrogen.
   3. Pierce sample lids with an 18 G needle and place in the lyophilizer chamber. Add paper towels for packing as needed.
   4. Fix lyophilizer chamber to lyophilizer and freeze dry overnight (at least -40 °C, 0.135 Torr)
7. Cation exchange column
   1. Resuspend desiccated samples in up to 400 μL of 8 M urea, 2% CHAPS solution (or, if pooling samples, resuspend to a max of (400/n) μL, where n = the number of samples in desired pool. Use as little of the detergent solution as possible.
   2. Equilibrate the cation exchange (IEX) column with 400 μL of 8 M urea, 2% CHAPS solution. Spin for 5 min at 2,000 x g at room temperature.
   3. Load 400 μL of sample/pooled samples into the IEX column. Spin for 5 min at 2,000 x g.
   4. Wash 3x with 400 μL of 8 M urea, 2% CHAPS solution. Spin for 5 min at 2,000 x g everytime.
   5. Elute 3x with 400 μL of 0.2 M NaCl. Spin for 5 min at 2,000 x g each. This is the low-affinity fraction - this can be retained for quality control purposes if desired.
   6. Elute 3x with 400 μL of 2.7 M (16%) NaCl. Spin for 5 min at 2,000 x g each. This fraction will contain the isolated glycosaminoglycans of interest - keep all of it!
   7. To desalt each eluted fraction, add methanol up to 80 vol% and incubate at 4 °C overnight. Spin each sample for 5 min at 2,000 x g. Recover the solid residue as this is dried de-salted glycosaminoglycan.
      NOTE: Alternatively, skip this step and proceed to step 1.8 to de-salt the eluate without methanol.
8. Sample desalting and concentration
   1. If necessary, pool eluted fractions (from 1.7.6) into appropriate experimental groups (e.g., by biological replicate, or experimental group).
   2. Place total sample volume into a 500 μL centrifugal filter column with a MWCO of 3,000 Da.
   3. Spin for 30 min at 14,000 x g at room temperature. Repeat as needed, if the desired sample volume exceeds capacity of the centrifugal filter column.
   4. Wash each column 3x with 400 μL deionized, filtered water. Discard flow through. Proceed directly to step 1.9.
9. Chondroitin digestion
   NOTE: The purpose of this step is to remove GAGs not of interest to the end user. In this case, chondroitinase is used to remove chondroitin. In the tissues used for generating Representative Results (broncho-alveolar lavage fluid and whole lung), digesting chondroitin sulfate leaves heparan sulfate as the primary residual GAG. End users may need to add additional digestion steps depending on their experimental aims. 
   1. Load 350 µL of digestion buffer (50 mM ammonium acetate with 2 mM calcium chloride adjusted to pH 7.0) to the centrifugal filter column without touching the membrane.
   2. Add 5 µL of recombinant chondroitinase ABC.
   3. Place the samples tube into a 37 °C oven and incubate for 1 h.
   4. Turn the column over and place into appropriately labeled collection tubes. Spin for 1 min at 2000 x g.
   5. Heat samples to 80 °C for 15-20 min to inactivate chondroitinase ABC.
10. Sample desalting and concentration
    1. If necessary, pool chondroitin digested samples from step 1.9.5 into appropriate experimental groups (e.g., by biological replicate, or experimental group)
    2. Place the total sample volume into a 500 μL centrifugal filter column with a MWCO of 3,000 Da.
    3. Spin for 30 min at 14,000 x g at room temperature. Repeat as needed if desired sample volume exceeds capacity of the centrifugal filter column.
    4. Wash each column 3x with 400 μL deionized, filtered water. Discard the flow through.
    5. Invert filter and spin for 1 min at 2,000 x g in fresh, appropriately labeled collection tubes. Freeze at -80 °C or proceed to next step.
11. Desiccation
    NOTE: In this step, desiccation is necessary so that samples can be resuspended in the smallest possible quantity of water and running buffer.
    1. Either place the chondroitin digested samples from step 1.10.5 directly in a rotational vacuum concentrator overnight or lyophilize as follows:
    2. Freeze samples thoroughly either overnight in -80 °C or by dipping in liquid nitrogen.
    3. Pierce sample lids with an 18 G needle and place in the lyophilizer chamber. Add paper towels for packing as needed.
    4. Fix lyophilizer chamber to lyophilizer and freeze dry overnight (at least 40 °C, 0.135 Torr)

2. Polyacrylamide gel electrophoresis of isolated and purified glycosaminoglycans

1. Prepare solutions necessary for polyacrylamide gel electrophoresis (PAGE) in advance (Table 1).
   NOTE: Select the percent acrylamide of resolving gel solution depending on the size of the glycosaminoglycans expected to be in the sample. 15% is recommended for resolving larger fragments (greater than 30 disaccharide subunits in length); 22% for smaller fragments (<20 disaccharide subunits in length).
2. Place empty cassette into the PAGE tank. Cast the resolving gel as follows: In 15 mL tube, mix 10 mL of resolving gel solution, 60 μL of 10% ammonium persulfate (must be freshly prepared), and 10 μL of TEMED (add TEMED last). Invert the tube gently 2-3x. Use pipette to quickly add the above 10 mL solution to cassette. Overlay with 2 mL of deionized, filtered water and allow the resolving gel to polymerize for 30 min.
   NOTE: The following PAGE protocol has been optimized for a vertical PAGE system using 13.3 x 8.7cm (width x length) 1.0mm thick casting cassettes with a total volume of approximately 12mL. Other cassette systems can be used but may require optimization by the end-user.
3. After the resolving gel has fully polymerized, discard the overlaid water and cast the stacking gel as follows: in a 15 mL tube, mix 3 mL of the stacking gel solution, 90 μL of 10% ammonium persulfate (must be freshly prepared), 3 μL of TEMED (add TEMED last).
4. Invert the tube gently 2-3x. Use a pipette to quickly add the stacking gel solution over the solidified resolving gel; fill cassette to brim. Fully insert comb included with the set up. Allow the stacking gel to polymerize for 30 min.
5. Once the gel has polymerized, ensure the tape strip is removed from the bottom of the cassette, and place the cassette back into the PAGE tank assembly.
6. Fill the upper and lower chambers with upper and lower chamber buffer, respectively.
7. Dissolve the dried samples from step 1.11.4 in the minimum necessary volume of deionized, filtered water (at most, 50% of the volume of the wells in the PAGE gel). Mix 1:1 with sample loading buffer. Load the samples and the HS oligosaccharide "ladders" (see Table 1) into the gel.
8. Pre-run the gel for 5 min at 100 V. Then run the gel at 200 V for 20-25 min (for a 15% polyacrylamide resolving gel), 40-50 min (for an 18% polyacrylamide resolving gel), 90-100 min (for 22% polyacrylamide resolving gel).
   NOTE: Some optimization of the 200 V run time may be necessary. Phenol red migrates ahead of heparin oligosaccharides that are 2 polymer subunits in length (i.e., degree of polymerization 2, or dp2); bromophenol blue migrates ahead of dp10-dp14. Best results are obtained when the voltage is applied such that the phenol red band migrates almost, but not quite, to the bottom of the gel. Adjust run time accordingly.

3. Silver staining protocol

1. Prepare all solutions necessary for silver staining in advance (Table 2).
   NOTE: Do not directly touch the PAGE gel until it has been stained, developed, and placed in stop solution. Instead, manipulate the gel using clean plastic or glass tools. Directly handling the gel will result in finger-print distortions and other visible artifacts on the gel after staining.  
2. Once the run is completed, disassemble cassette and extract gel in a clean, medium-large container filled with deionized, filtered water.
   NOTE: To avoid directly handling the gel, use pipette tip or other plastic object to gently peel the gel away from the cassette while submerged in water. Gel may be fragile - handle carefully.
   1. Discard the water. Stain the gel in Alcian blue staining solution for 5 min.
   2. Discard Alcian blue stain. Quickly rinse/wash 2-3x with deionized, filtered water until most of the Alcian blue staining solution has been removed.
   3. Allow to de-stain in deionized, filtered water overnight on rocker. Ensure that there is ample volume of deionized, filtered water to ensure any residual stain is fully washed off the gel overnight.
   4. Wash gel in 50% methanol (40 min total, change solution 2-3x).
   5. Wash gel in deionized, filtered water for 30 min. Discard water and repeat 3 more time for a total of 2 h, replacing the water each time.
   6. In a fresh, clean container, stain the gel for 30 min in silver nitrate staining solution.
   7. Quickly rinse/wash 2-3x in deionized, filtered water to fully remove the silver staining solution.
   8. Wash for 30 min in deionized, filtered water. Discard water and repeat 2x for a total of 90 min, replacing the water bath each time.
   9. Discard water and add developing solution.
   10. Once developing solution is added, carefully observe the gel and watch for the appearance of bands. Depending on the quality of the stain and the mass of the sample loaded, development can take anywhere from a few seconds to several minutes.
   11. As soon as the desired bands are visible, immediately discard developing solution and wash briefly with stop solution.
   12. Discard the stop solution wash and replace with fresh stop solution. Allow to soak for 1 h on a rocker or shaker.
   13. Wash in deionized, filtered water overnight (however, the gel can be imaged immediately after stop solution wash).

Wyniki

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...

Dyskusje

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...

Materiały

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