This work was supported by National Institutes of Health (NIH) Common Fund for Glycoscience grant U01CA241953. MJP was supported by the Chemistry-Biology Interface Program at Johns Hopkins (T32GM080189).

Tissue collection was performed under conditions authorized by the Johns Hopkins Animal Care and Use Committee.
1. Small scale ganglioside extraction and partial purification
CAUTION: Use appropriate ventilation when working with volatile and toxic solvents. Avoid plastic throughout; solvents will extract chemical components from many plastics that interfere with subsequent analyses. Polytetrafluoroethylene (PTFE) is an exception; PTFE-lined closures should be used to cap glass storage vials.
<ol>
	<li>Extraction
	<ol>
		<li>Weigh a single fresh or thawed mouse brain (or sagittal half-brain, ~ 0.2-0.5 g) and place in a Potter-Elvehjem homogenizer prechilled in a bucket of ice. 
		NOTE: Previously frozen brains can be used after thawing at 0-4 &#176;C.</li>
		<li>Add 4.1 mL per g tissue wet weight of water and homogenize with 10 strokes. 
		NOTE: Accurate solvent ratios are key to optimal extraction and partition, the goal is chloroform-methanol-aqueous (4:8:3) assuming brain tissue is 80% aqueous.</li>
		<li>Add 13 mL per g tissue wet weight of methanol, shift to ambient temperature (22 &#176;C) and mix. 
		&#8203;NOTE: The solution will appear cloudy. Addition of methanol at this step, without chloroform, optimizes protein precipitation. All subsequent steps are at ambient temperature (22 &#176;C).</li>
		<li>Transfer to a thick-walled glass screw-capped tube with a PTFE-lined screw cap at ambient temperature (22 &#176;C) and mix thoroughly. Add 6.5 mL per g tissue wet weight of chloroform, cap, and mix thoroughly. Centrifuge at 450 x g for 15 min. Transfer the clear supernatant to a fresh screw-capped tube and measure the volume, &#34;recovered extract volume&#34;.</li>
	</ol>
	</li>
	<li>Partition
	<ol>
		<li>Add 0.173x &#34;recovered extract volume&#34; of water to the clear supernatant, cap, vortex vigorously, and centrifuge as described in step 1.1.4. 
		NOTE: The goal is to have chloroform-methanol-aqueous in the ratio 4:8:5.6. The mixture will be cloudy and resolve into two phases: an upper aqueous-rich phase and a lower chloroform-rich phase at ~ 4:1 ratio. Wait for 60 min or centrifuge at 450 x g for 15 min for complete phase separation.</li>
		<li>Transfer the upper phase, which contains the gangliosides, into a fresh glass tube with a PTFE-lined screw cap.</li>
	</ol>
	</li>
	<li>Reverse phase cartridge chromatography
	<ol>
		<li>Using a 5 mL glass syringe, wash a tC18 solid phase extraction cartridge (400 mg) with 3 mL of methanol, then 3 mL of chloroform-methanol-water (2:43:55). Load the upper phase from step 1.2.2 onto the tC18 cartridge using&#160;the same glass syringe, collect the flow-through and reload it onto the column to optimize adsorption.</li>
		<li>Using the glass syringe, wash the cartridge with 3 mL of chloroform-methanol-water (2:43:55) then 3 mL of methanol-water (1:1).</li>
		<li>Elute the gangliosides with 3 mL of methanol into a fresh screw-capped tube. Evaporate to dryness under a gentle stream of nitrogen at &#8804; 45 &#176;C. Dissolve in methanol at 1 mL per g of original tissue wet weight.</li>
	</ol>
	</li>
</ol>
2. Large scale ganglioside extraction and purification
CAUTION: When working with volatile solvents, use explosion resistant blenders. Do not use plastics except PTFE. Tetrahydrofuran, chloroform, and ethyl ether are toxic volatile organic compounds. Work in a fume hood with protective gloves and safety goggles.
<ol>
	<li>Extraction
	<ol>
		<li>Thaw frozen bovine brain at 4 &#176;C for several hours. Dissect the grey matter from meninges and white matter. 
		NOTE: The following procedure is described for 100 &#177; 20 g of isolated brain grey matter and is scalable.</li>
		<li>Place 100 g of brain grey matter in a blender and add 1 mL per g brain wet weight of chilled 10 mM potassium phosphate buffer pH 6.8. Homogenize on low for 20 s. Add 8 mL tetrahydrofuran per g brain wet weight and homogenize on low for 10 s. Decant into glass centrifuge bottles and centrifuge at 5,000 x g for 15 min at ambient temperature (22 &#176;C).</li>
		<li>Collect the supernatant, measure its volume, and transfer to a glass separatory funnel. Add 0.3 mL of ethyl ether per mL of the supernatant. Shake vigorously, then allow to sit undisturbed for 30 min during which two phases, an upper ether phase and a lower aqueous phase, separate. Collect the lower phase, which contains the gangliosides, into a glass bottle with a PTFE-lined cap.</li>
		<li>To the upper (ether) phase remaining in the separatory funnel, add 0.1 mL water per mL of original supernatant (step 2.1.3). Shake vigorously, allow phases to separate, collect the lower (aqueous) phase and combine with the previous lower phase. Evaporate the combined lower phases to a dry powder and weigh.</li>
	</ol>
	</li>
	<li>Saponification
	<ol>
		<li>Add 10 mL of 100 mM aqueous NaOH per g powder in a sealed tube. Mix and incubate at 37 &#176;C for 3 h. Allow to cool and adjust to pH 4.5 by dropwise addition of 100 mM aqueous HCl. Measure the volume and transfer to a glass separatory funnel. 
		NOTE: Sialic acids are acid labile; avoid acidification below pH 4.5.</li>
		<li>Based on the aqueous volume, add 2.67 volumes of methanol, mix gently, then add 1.33 volumes of chloroform to create a single-phase solution of chloroform-methanol-aqueous (4:8:3). Mix well.</li>
		<li>Based on the original aqueous volume, add 2.6 volumes of water to bring the mixture to chloroform-methanol-aqueous to a ratio of 4:8:5.6. Shake vigorously, then allow to sit undisturbed to separate two phases, a polar upper phase containing the gangliosides and a nonpolar lower phase. Collect the upper phase in a glass bottle with a PTFE-lined cap. 
		NOTE: Non-sialylated lipids will not appear on thin layer chromatography (TLC) plates stained using resorcinol but will appear when using a p-anisaldehyde stain. The purpose of this saponification is to remove the O-acetylated compounds such as phospholipids.</li>
	</ol>
	</li>
	<li>Reverse phase chromatography
	<ol>
		<li>Pre-wash a large scale (10 g) tC18 solid phase extraction cartridge by passing 50 mL of each of the following three solvents through the column using vacuum or pressure (&#60;1 min each wash): methanol, methanol-water (1:1), then chloroform-methanol-water (2:43:55). Load the upper phase from step 2.2.3 onto the column by vacuum or pressure, collect the flow through, reload, and collect the flow through.</li>
		<li>Wash the column with 30 mL of chloroform-methanol-water (2:43:55), then 30 mL methanol-water (1:1), then elute the gangliosides with 50 mL methanol, and again with 10 mL methanol, collecting each wash and each elution separately. Using TLC (below) confirm that ganglioside is absent from the flow through and washes and eluted in the first elution (50 mL methanol). Evaporate the eluted gangliosides to a dry powder and weigh. 
		NOTE: The purpose of tC18 chromatography is to separate gangliosides from both less and more polar contaminants. Mixed brain ganglioside yield after saponification is ~ 120 mg per g dry brain extract (step 2.1.4). Appearance of gangliosides by TLC in the flow through or washes indicates the solid phase extraction column was saturated. After methanol elution, the column may be further eluted with chloroform-methanol (1:1) to capture less polar lipids.</li>
	</ol>
	</li>
	<li>HPLC purification of individual gangliosides
	<ol>
		<li>Prepare HPLC Solvent A: acetonitrile-5 mM aqueous sodium phosphate buffer pH 5.6 (83:17) and Solvent B: acetonitrile-20 mM sodium phosphate buffer, pH 5.6 (1:1). Degas both solvents for 5 min.</li>
		<li>Pre-equilibrate an HPLC column (20 x 250 mm column packed with amine bonded (NH2) silica spheres, 5 &#181;m diameter, 100 &#197; pore size) with 100% Solvent A for 20 min at 5 mL/min. Set a UV HPLC column effluent detector to 215 nm.</li>
		<li>Dissolve the ganglioside powder from the reverse phase eluate in water at 5 mg/mL. Inject 0.5 mL of the ganglioside mixture onto the HPLC and run the solvent gradient (Table 1) at 5 mL/min, collecting fractions. Gangliosides will appear as A215 peaks with retention times (major brain gangliosides) of 25-70 min: GM1 &#8776; 28 min; GD1a &#8776; 38 min; GD1b &#8776; 46 min; GT1b &#8776; 65 min. Re-equilibrate 20 min with Solvent A after each run. Analyze fractions by thin-layer chromatography.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>%A</td>
			<td>%B</td>
		</tr>
		<tr>
			<td>0</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>7</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>12</td>
			<td>63</td>
			<td>37</td>
		</tr>
		<tr>
			<td>82</td>
			<td>54</td>
			<td>46</td>
		</tr>
		<tr>
			<td>82.01</td>
			<td>0</td>
			<td>100</td>
		</tr>
		<tr>
			<td>92</td>
			<td>0</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
Table 1: Solvent gradient for HPLC.
3. Thin layer chromatography (TLC) analysis of gangliosides
CAUTION: Chloroform is a toxic volatile organic compound. Work in a fume hood with protective gloves and safety goggles.
<ol>
	<li>Running solvent and TLC plate preparation
	<ol>
		<li>Prepare a running solvent of chloroform-methanol-aqueous 0.25% KCl (60:35:8 by volume). Pour into a 10 cm x 10 cm glass TLC chamber with a stainless-steel cover so that the solvent depth is ~ 0.5 cm. Cover and allow to equilibrate in an area free of air currents for &#62;10 min. 
		NOTE:To isolate the TLC chamber from air currents, an acrylic 5-sided box can be constructed or purchased&#160; (Figure 2). Do not use solvent-saturated filter paper inside the chamber.</li>
		<li>Place a 10 cm x 10 cm or 5 cm x 10 cm silica gel coated glass-backed high performance TLC plate in a drying oven at 125 &#176;C for 10 min. Allow to cool. Use a dulled #2 pencil to draw 5-mm spotting lines with 2-mm separations along a line 1 cm above bottom of the plate and at least 1 cm from either side (Figure 3). Avoid disturbing the silica layer while marking.</li>
		<li>Prepare a standard mix of pure gangliosides in methanol containing 100 &#181;M GM1, 50 &#181;M each of GD1a and GD1b and 33 &#181;M of GT1b. 
		NOTE: This mixture contains 100 pmol of ganglioside sialic acid per &#181;L for each of the four gangliosides, a quantity that provides a strong colorimetric signal by resorcinol staining, which is sialic acid dependent.</li>
	</ol>
	</li>
	<li>Ganglioside resolution
	<ol>
		<li>Wash a 10-&#181;L Hamilton syringe with a beveled needle with methanol. Draw 1 &#181;L methanol into a glass syringe to fill the needle dead volume and then 1 &#181;L of sample or standard. Spot the sample evenly onto the 5-mm premarked lines until &#60;1 &#181;L of solvent (methanol) remains in the syringe. Allow the plate to dry at ambient temperature (22 &#176;C) after all samples are spotted. 
		NOTE: Wash the syringe with methanol between sample loading. An unheated air blower set at low can be used to accelerate drying.</li>
		<li>Place the spotted and dried plate into the preequilibrated TLC chamber with the bottom edge immersed in the running solvent and cover and protect from air currents (Figure 2). Allow the running solvent to advance up the plate by capillary action until the solvent front reaches within 1 cm of the top of the plate. Remove and mark the solvent front at the edge of the plate with a pencil. Allow the solvents to evaporate completely either undisturbed or under mild air flow.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62385/62385fig02.jpg" /> 
Figure 2: Ganglioside TLC equipment and set up. A twin trough chamber is filled to &#8776; 0.5 cm on both sides with running solvent. The plate is placed against one side with the origin end immersed in the running buffer. The chamber is covered with an acrylic box to avoid air currents. Panel A, side view prior to plate insertion. The solvent level is visible a few mm above the chamber bottom; Panel B, front view during development. The solvent front is visible at about 40% of the way up the plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62385/62385fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Ganglioside staining 
	CAUTION: Reagent stains are toxic. Hydrochloric acid is corrosive and toxic. Prepare and spray reagents in a fume hood with protective gloves and safety goggles. 
	NOTE: The plate can be imaged for qualitative image analysis or stored by removing the clamps and securing the cover plate in place with clear tape. Quantitative analysis can be performed by measuring densitometry of ganglioside standards spotted in adjacent lanes.
	<ol>
		<li>Prepare resorcinol spray reagent for the detection of gangliosides based on their sialic acids. Dissolve 6 g of resorcinol in 100 mL water for a 6% resorcinol stock. Dissolve 1 g of CuSO4 in 100 mL of water to make a 1% stock. To 64.7 mL of water add 5 mL of the 6% resorcinol stock, 0.31 mL of the 1% CuSO4 stock then slowly add 30 mL of concentrated HCl and stir gently. May be stored at 4 &#176;C for a month.</li>
		<li>In a chemical fume hood, place the TLC plate with resolved gangliosides, origin end up, in a cut-away cardboard box to protect the walls of the hood from acid spray. Place resorcinol spray reagent in a glass TLC sprayer, attach to a source of pressurized nitrogen, and lightly spray the plate diagonally in the vertical and horizontal directions. Spray the TLC sorbent surface uniformly, but lightly.</li>
		<li>Immediately cover the plate with a clean dry glass cover plate of the same dimensions and secure the cover plate in place with binder clips (Figure 3). Heat the covered plate at 125 &#176;C for 20 min. Gangliosides will appear dark purple against a white background. 
		NOTE: The plate should not appear wet when spraying is complete. Cover plates can be fashioned by scraping the sorbent from previously used TLC plates using a single-edge razor blade. 
		CAUTION: Silica powder is toxic to lungs. Use a mask and dispose silica in a sealed container.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62385/62385fig03.jpg" /> 
Figure 3: TLC plate of resolved mixed ganglioside. TLC plate of resolved mixed ganglioside standards (left lane) and purified mixed bovine grey matter gangliosides (right lane) after resorcinol staining and heating with glass cover plate clipped in place. Standard gangliosides (top to bottom) are GM3, GM2, GM1, GD3, GD1a, GD1b, GT1b and GQ1b. After cooling, the plate can be imaged and/or the cover plate taped in place for storage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62385/62385fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>General lipid staining for gangliosides and phospholipids. 
	&#8203;CAUTION: Sulfuric acid is toxic and corrosive. Addition of concentrated acid to ethanol is exothermic and must be done slowly. Prepare staining reagent in a fume hood with protective gloves and safety goggles.
	<ol>
		<li>Prepare p-anisaldehyde stain by slowly adding 15 mL of concentrated sulfuric acid to 500 mL of ethanol. Stir for 30 min to allow the solution to cool before proceeding. Add 15 mL p-anisaldehyde and stir gently. This may be stored at room temperature (22 &#176;C) up to six months.</li>
		<li>In a chemical fume hood, dip the TLC plate with resolved gangliosides, origin end&#160;down, into a beaker containing the p-anisaldehyde stain. Submerge to the running front for &#8805; 2 s. Remove TLC from stain and allow to drain. Heat the TLC plate on a hot plate at low temperature to develop. 
		NOTE: Lipids will appear dark against a purple background. Staining solution may be recovered for repeated use.</li>
	</ol>
	</li>
</ol>

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The authors claim no competing interests.

The methods for small and large scale ganglioside extraction and isolation reported here are not unique - there are many different solvent extraction and purification approaches that provide excellent results12. The methods reported here for small scale purification from brain, from Fredman and Svennerholm13, were shown to optimize recovery and have proven to be robust and straightforward over many years in our laboratory. Isolation and purification suitable for TLC and MS can be readily completed, from intact tissue to isolated gangliosides, in a few hours. MS can be performed on native purified gangliosides in the negative mode or after permethylation in the positive mode (Figure 7, e.g., see Sturgill et al.9). The yield is very consistent, &#8776; 1 &#181;mol ganglioside (&#8776; 2 &#181;mol ganglioside-bound sialic acid) per g fresh brain tissue for most mammals. The method for large scale extraction and partition from brain reported here, introduced by Tettamani et al.14, is selected to minimize the volume of ganglioside-containing solvent at the first partition (ether-tetrahydrofuran-water). This simplifies subsequent steps that can become cumbersome with techniques that generate large volumes of partitioned gangliosides at the first steps. The HPLC method described, from Gazzotti et al.15, has relatively high capacity and good resolution of the major brain gangliosides. 
The small-scale protocols described are well suited for both cells and tissues and are scalable. Since the final steps are reverse-phase capture, evaporation and redissolving in methanol, they can be applied at an arbitrary scale to small samples, such as cultured nerve cells. In this case, we scrape and homogenize cells into 1 mL of water for convenience and proceed with methanol and chloroform addition to generate the appropriate ratios for extraction and then partition. The final dried gangliosides after reverse phase can be redissolved in just a few microliters and analyzed by TLC and MS.
Attention to solvent ratios is critical to success in extraction and solvent partitioning steps for ganglioside isolation. For small scale extraction and partitioning, different chloroform-methanol-water ratios were tested and the ones that generated near quantitative ganglioside isolation are reported here13. Variations from the described ratios will diminish recovery and/or purification. Likewise, attention to solvent ratios in TLC developing solvents is critical. The chloroform-methanol-water ratios reported result in a single clear phase. Small variations may yield cloudy developing solutions which should not be used but can be clarified by dropwise addition of methanol with swirling. Although different extraction and partition solvents are common, solvent ratios should be carefully followed for each variation12. Alterations in TLC solvents are also common to optimize separation of specific gangliosides16. A relatively simple way to modify TLC migration of gangliosides is to change the aqueous phase of the solvent mixture. Using aqueous ammonium hydroxide or calcium chloride instead of potassium chloride alters the ganglioside salt form and relative TLC migration17. 
Whereas all vertebrate cells and tissues express gangliosides, the brain and nerve cells are unusual in the high amounts expressed. The protocols described here are applicable to other tissues and cells, but modifications may be required due to the lower abundance and potential contamination with other lipids. These methods as applied to human neutrophils6 and mouse adipose18 provide examples.

Gangliosides are defined as glycosphingolipids bearing one or more sialic acid residues1. They are expressed primarily at the cell surface with their hydrophobic ceramide lipid moiety embedded in the outer leaflet of the plasma membrane and their hydrophilic glycans extending into the extracellular space2. Although distributed widely in vertebrate cells and tissues, they are particularly abundant in the vertebrate brain3, where they were first discovered and named4.
The structures of ganglioside glycans vary and are the basis for their nomenclature (Figure 1). Ganglioside glycans are comprised of a neutral sugar core bearing different numbers and distributions of sialic acids. The smallest ganglioside, GM4, has only two sugars (sialic acid bound to galactose)5. Larger naturally occurring gangliosides may contain well over a dozen total sugars6 or up to seven sialic acids on a single neutral core7. Their ceramide lipid moieties also vary, having different sphingosine lengths and a variety of fatty acid amides. In the vertebrate brain four ganglioside species, GM1, GD1a, GD1b, and GT1b predominate. Ganglioside expression is developmentally regulated, tissue specific, and cell type specific.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62385/62385fig01.jpg" /> 
Figure 1: Major brain gangliosides and their biosynthetic precursors. Structures are shown using Symbol Nomenclature for Glycans11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62385/62385fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Gangliosides function at the molecular level by engaging and modulating proteins in their own membranes (cis regulation) or by engaging glycan binding proteins in the extracellular milieu, including bacterial toxins and lectins on other cells (trans recognition)3. Specific binding of gangliosides to regulatory proteins and/or self-association with other molecules into lipid rafts results in changes in cell behavior that impact nervous system structure and function, cancer progression, metabolism, inflammation, neuronal proteinopathies, and infectious diseases8. Because of their diverse cellular roles, methods for their isolation and analysis can provide enhanced insights into the regulation of physiological and pathological processes. Here, validated methods for rapid small-scale extraction and analysis, and preparative scale isolation of gangliosides from brain are provided. Opportunities and challenges for application to other tissues are discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine brain, stripped</td>
			<td>PelFreez</td>
			<td>57105-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ganglioside standards</td>
			<td>Matreya</td>
			<td>GM1, 1061; GD1a, 1062; GD1b, 1501; GT1b, 1063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottle with PTFE-lined cap</td>
			<td>Fisher Scientific</td>
			<td>02-911-739</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass centrifuge bottle</td>
			<td>Fisher Scientific</td>
			<td>05-586B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass culture tubes, 16 x 125 mm</td>
			<td>VWR</td>
			<td>60825-430</td>
			<td>for collecting HPLC fractions</td>
		</tr>
		<tr>
			<td>Glass separatory funnel (2 L)</td>
			<td>Pyrex</td>
			<td>6400-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection syringe - Hamilton 1750 gastight 500 &#181;l</td>
			<td>Hamilton</td>
			<td>81265</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Anisaldehyde, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;A88107</td>
			<td></td>
		</tr>
		<tr>
			<td>Potter-Elvhjem Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>08-414-14A</td>
			<td>Choose appropriate volume option</td>
		</tr>
		<tr>
			<td>Reprosil 100 NH2 10&#181;m 5x4mm guard columns</td>
			<td>Analytics-Shop</td>
			<td>AAVRS1N-100540-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Reprospher 100 NH2, 5 &#956;m, 250 mm x 20 mm HPLC column</td>
			<td>Analytics-Shop</td>
			<td>custom packed</td>
			<td>other sizes available</td>
		</tr>
		<tr>
			<td>Resorcinol</td>
			<td>Sigma-Aldrich</td>
			<td>30752-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Buchi</td>
			<td>R-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample loop for Model 7725 Injector (5 ml)</td>
			<td>Sigma-Aldrich</td>
			<td>57632</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak tC18 Cartidges Vac 35 cc (10 g)</td>
			<td>Waters</td>
			<td>WAT043350</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak tC18 Plus Short Cartridge, 400 mg</td>
			<td>Waters</td>
			<td>WAT036810</td>
			<td></td>
		</tr>
		<tr>
			<td>Spotting syringe - Hamilton 701N 10 &#181;l</td>
			<td>Hamilton</td>
			<td>80300</td>
			<td></td>
		</tr>
		<tr>
			<td>Thick-walled 13-mm diameter test tubes with PFTE lined caps</td>
			<td>Fisher Scientific</td>
			<td>14-933A</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded 2-ml vials with PFTE lined caps</td>
			<td>Fisher Scientific</td>
			<td>14-955-323</td>
			<td>For ganglioside storage</td>
		</tr>
		<tr>
			<td>TLC plates, HPTLC Silica gel 60 F254 Multiformat</td>
			<td>Fisher Scientific</td>
			<td>M1056350001</td>
			<td>Fluorescence impregnation (F254) stabilizes the sorbent surface</td>
		</tr>
		<tr>
			<td>TLC reagent sprayer</td>
			<td>Fisher Scientific</td>
			<td>05-723-26A</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC running chamber for 10 x 10 cm plates</td>
			<td>Camag</td>
			<td>22.5155</td>
			<td></td>
		</tr>
		<tr>
			<td>Waring 1-Liter Stainless Steal Explosion Resistant Blender</td>
			<td>Waring</td>
			<td>E8520</td>
			<td></td>
		</tr>
	</tbody>
</table>

ganglioside extraction, purification and profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

The methods described in section 1 (small scale) provide gangliosides at sufficient quantity and purity for qualitative and quantitative determination of major brain gangliosides. Recovery from mouse brain is ~ 1 &#181;mol ganglioside per g brain wet weight (1 nmol/&#181;L) when prepared as described. TLC resolution of 1 &#181;L (1 nmol) using section 3 provides ample material for resorcinol detection and resolves all of the major brain gangliosides as shown for wild type and genetically modified mice in Figure 4. Although mixed gangliosides prepared using section 1 are not free of other major lipids, gangliosides are of sufficient purity for mass spectrometric (MS) determination as shown in Figure 5, either as native purified gangliosides in negative mode or after permethylation in positive mode9. Since section 1 avoids alkaline hydrolysis to remove phospholipids, it retains alkali-sensitive natural modifications, such as O-acetylated sialic acids (see GT1b-OAc, Figure 4)9.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62385/62385fig04.jpg" /> 
Figure 4: TLC of mouse brain gangliosides. TLC of mouse brain gangliosides from wild-type (WT) and St3gal single and double-null mice purified as in Protocol 1. This figure has been modified from Sturgill et al, 20129. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62385/62385fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62385/62385fig05.jpg" /> 
Figure 5: MS of permethylated wild-type and St3gal2/3-double-null mouse brain gangliosides purified as in section 1. Note that&#160;GD1a and GD1b resolve by TLC (Figure 4) but have the same mass so are not distinguishable by one-dimensional MS. This figure has been modified from Sturgill et al, 20129. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62385/62385fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Large scale purification (section 2) includes extraction, saponification (to remove phospholipids) and HPLC resolution to provide purified major brain gangliosides GM1, GD1a, GD1b and GT1b suitable for biological experiments and for further chemical and enzymatic modifications. An exemplary HPLC profile and subsequent TLC analysis are shown in Figure 6. Alkali treatment (saponification) is necessary in this protocol to hydrolyze and remove contaminating phospholipids (Figure 7) but will also hydrolyze natural modifications of gangliosides, such as O-acetylated sialic acids, which may be important in some contexts10. For these applications, alternate effective methods for removal of phospholipids from isolated gangliosides have been published6.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62385/62385fig06.jpg" /> 
Figure 6: Representative HPLC of bovine brain gangliosides. The elution gradient (%B, dotted line) is overlaid on the absorbance (A215, solid line) for the first 75 min of the cycle. Peaks (A215) were collected (numbers in brackets) and subjected to TLC as in Protocol 3. Lane numbers refer to the peak numbers on the chromatogram. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62385/62385fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62385/62385fig07.jpg" /> 
Figure 7:&#160;TLC plate of resolved mixed ganglioside. TLC plate of resolved mixed gangliosides standards (lane 1) along with post-saponification partitioned gangliosides (lane 2) and released fatty acids (lane 3). Lipids, including gangliosides, are detected with p-anisaldehyde stain. Standard gangliosides (top to bottom) are GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62385/62385fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ganglioside Extraction, Purification and Profiling at JoVE.com

Ganglioside Extraction, Purification and Profiling

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

ganglioside-extraction-purification-and-profiling

Nanyang Technological University

Research

JoVE Journal

Biochemistry

4.3K Views.  Johns Hopkins University School of Medicine. Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Video: Ganglioside Extraction, Purification and Profiling

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Extraction and Analysis of Taiwanese Green Propolis

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...