Product Description

Preparation of Glycans

Pre-001. Preparation of mixed N-Glycans. The starting material for this procedure can be purified protein, cell extract or tissue. Protein is solubilized and denatured with SDS/2-ME. NP-40 is added to "buffer" the SDS. PNGase F is added and the mixture incubated overnight at 37oC. The mixture is applied to a Sep-Pak C18 cartridge equilibrated in water. Proteins and the majority of detergent binds to the resin. The run through and water washes are applied to a porous graphitized carbon (PGC) cartridge. Under aqueous conditions, oligosaccharides bind to the PGC cartridge, while salts and buffers pass through unretarded. Carbohydrates are eluted with 30% acetonitrile, 0.1% TFA in water while any residual detergent remains bound to the cartridge. The resulting desalted, N-glycans are dried and ready for further analysis.

Pre-002. Preparation of Mixed O-Glycans

The starting material for this procedure can be purified protein, cell extract or tissue. The sample is incubated with 0.1 M NaOH/1M NaBH4 at 45oC overnight to release O-glycans by way of alkaline induce -elimination. The sample is passed through a cartridge packed with Dowex 50 (H+ form) to remove peptide and Na+. The flow through is collected and lyophilized. Repeated addition of methanol/acetic acid to form volatile methylborates followed by drying under a stream of nitrogen yields desalted, dried, reduced O-glycans ready for further analyis.

Pre-003. Preparation of Mixed Glycolipid-derived Glycans

The starting material for this procedure can either be purified glycolipids or crude lipid extracts, e.g., Pre-004. Lipid mixtures are treated with endoglycoceramidase to release the glycan from the lipid. The mixture is passed over a Sep-Pak C18 cartridge to remove enzyme and lipid and the run-through is applied to a PGC cartridge. Under aqueous conditions, oligosaccharides bind to the PGC cartridge, while salts and buffers pass through unretarded. Carbohydrates are eluted with 30% acetonitrile, 0.1% TFA in water while any residual detergent remains bound to the cartridge. The resulting desalted, glycolipid-derived glycans are dried and ready for further analysis.

Pre-004. Preparation of Total Lipid Extracts

The starting material for this procedure is typically tissues. Tissues are sequentially extracted with chloroform:methanol, twice with each of the following ratios 2:1, 1:1 and 1:2. The pooled extracts are dried and resuspended in chloroform:methanol 2:1.

Prep-005. Preparation of Cellular Metabolites: TCA Extraction

The starting material for this procedure is typically tissue or cell pellets. Freshly isolated material is preferable. Samples are treated with TCA (5-10% w/v final). The precipitated macromolecules are removed by centrifugation. The supernatant is treated with an equal volume of Freon:trictylamine (3:1) to extract the TCA and the aqueous phase is stored frozen or lyophilized.

Modification of Glycans

Mod-001. Reduction of Glycans. The starting material for this procedure can be any glycan or glycan mixture. Glycans are incubated in the presence of sodium borohydride to reduce the reducing terminal. The mixture is passed over Dowex 50 (H+ form) to remove Na+. The flow through is collected and lyophilized. Repeated addition of methanol/acetic acid to form volatile methylborates followed by drying under a stream of nitrogen yields desalted, dried, reduced glycans ready for further analyis.

Mod-002. Permethylation of Glycans

Starting material for this procedure is dried, desalted glycans. Glycans are resuspended in a slurry of NaOH in DMSO and methyl iodide is added to methylate all exposed hydroxyl groups. The resulting derivatized saccharides are bound to a Sep-Pak C18 cartridge and eluted with increasing concentrations of acetonitrile. The resulting desalted permethylated glycans are dried and ready for analysis by either MALDI-TOF mass spectrometry or ESI-IT mass spectrometry.

Mod-003. Exoglycosidase Digestion

Starting material for this procedure is dried, desalted glycans. Glycans are resuspended in buffer appropriate for each digestion. One or more exoglycosidase(s) can be used to treat the sample. Following digestion, enzyme and buffer salts are removed by either solid phase extraction or gel filtration depending on the nature of the sample and the desalted sample is lyophilized.

Mod-004. Glycosaminoglycan Lyase Digestion

The starting material for this procedure is dried, desalted glycosaminoglycan. Glycosaminoglycans are dissolved in buffer appropriate for the lyase(s) to be used. Following digestion, the mixture is fractionated using a 10,000 NMWCO filter to remove enzyme and undigested glycosaminoglycan chains.

Mod-005. Mild Acid Treatment to Release Sialic Acids

The starting material for this procedure is typically isolated glycan, although glycopeptides and intact proteins may also be deemed appropriate depending on the nature of the sample. Sample is dissolved in a final concentration of 2 M HOAc and heated to 80oC for 3 hours. Acetic acid is removed by drying.

Profiling Methods Pro-001. HPAEC-PAD Profile of N-Glycans

The starting material for this procedure is a mixture of desalted N-glycans, e.g., Pre-001. Reduced N-glycans can be similarly analyzed. The sample is dissolved in water and profiled by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) using either a Dionex Carbo-Pak PA-1 or PA-100 column. Increasing concentrations of NaOH and NAOAc effect elution. Species elute in order of increasing negative charge. Elution time is also affected by the size of the oligosaccharide, the nature of the glycosidic linkages and the presence of fucose residues. Detection limits of ~10 pMoles/peak are typical. Control analysis consists of profiling a standard mixture of fetuin N-glycans. Fractions may be collected for an additional charge. Scintillation counting is also available. The data presented to the investigator consists of the HPLC data trace and a peak table including peak retention times and areas. Powerpoint formatted data are available for an additional charge.

Pro-002. HPAEC-PAD Profile of O-Glycans

The starting material for this procedure is a mixture of desalted (generally reduced) O-glycans, e.g., Pre-002. The sample is dissolved in water and profiled by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) using either a Dionex Carbo-Pak PA-1 or PA-100 column. Increasing concentrations of NaOH and NAOAc effect elution. Species elute in order of increasing negative charge. Elution time is also affected by the size of the oligosaccharide, the nature of the glycosidic linkages and the presence of fucose residues. Gradient conditions differ from those used for N-glycans. Detection limits of ~10 pMoles/peak are typical. Control analysis consists of profiling a standard mixture of either bovine submaxillary gland O-glycans, or milk oligosaccharides. Fractions may be collected for an additional charge. Scintillation counting is also available. The data presented to the investigator consists of the HPLC data trace and a peak table including peak retention times and areas. Powerpoint formatted data are available for an additional charge.

Pro-003. HPAEC-PAD Profile of O-Glycan Alditols

The starting material for this procedure is a mixture of desalted, reduced O-glycans, e.g., Pre-002. The sample is dissolved in water and profiled by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) using a Dionex Carbo-Pak MA-1 column. This procedure is preffered over Pro-002 for small, neutral alditols. Increasing concentrations of NaOH and NAOAc effect elution. Species elute in order of increasing negative charge and size. Elution time is also affected by the nature of the glycosidic linkages and the presence of fucose residues. Detection limits of ~10 pMoles/peak are typical. Control analysis consists of profiling a standard mixture of either reduced bovine submaxillary gland O-glycans, or reduced milk oligosaccharides. Fractions may be collected for an additional charge. Scintillation counting is also available. The data presented to the investigator consists of the HPLC data trace and a peak table including peak retention times and areas. Powerpoint formatted data are available for an additional charge.

Pro-004. HPAEC-PAD Profile of Glycolipid-derived Glycans

The starting material for this procedure is desalted glycans enzymatically released from glycolipids, e.g., Pre-003. The sample is dissolved in water and profiled by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) using either a Dionex Carbo-Pak PA-1 or PA-100 column. Increasing concentrations of NaOH and NAOAc effect elution. Species elute in order of increasing negative charge. Elution time is also affected by the size of the oligosaccharide, the nature of the glycosidic linkages and the presence of fucose residues. Detection limits of ~10 pMoles/peak are typical. Control analysis consists of profiling a standard mixture of mixed ganglioside-derived glycans. Fractions may be collected for an additional charge. Scintillation counting is also available. The data presented to the investigator consists of the HPLC data trace and a peak table including peak retention times and areas. Powerpoint formatted data are available for an additional charge.

Pro-005. Anion Exchange Profile of Cellular Metabolites with UV Detection

The starting material for this procedure is typically a TCA extract from cells or tissues, e.g., Pre-005. The mixture is profiled by anion exchange chromatography using a Dionex PA-1 column equilibrated in either water or 10mM NaOH. Elution is efffected by a gradient of increasing NaOAc concentration. UV detection is typically performed at 260 nm. The dominant species present in this analysis are typically nucleotide phosphates. Sugar nucleotides are well resolved using a combination of the two gradient systems described above, however true estimates of sugar nucleotide concentrations may first require the removal of nucleotide phosphates. Detection limits of ~1 nMoles/peak are typical. Control analysis consists of profiling a standard mixture of sugar nucleotides. Fractions may be collected for an additional charge. Scintillation counting is also available. The data presented to the investigator consists of the HPLC data trace and a peak table including peak retention times and areas. Peaks co-migrating with known standards will be so marked. Powerpoint formatted data are available for an additional charge.

Pro-006. Profile of Glycosaminoglycan-derived Disaccharides by Anion Exchange HPLC with UV and Fluorescent Detection

The starting material for this procedure is typically a mixture of disaccharides obtaining by the action of lyases. This method is prefered for heparin- and heparan sulfate-derived disaccharides. Elution is effected with a gradient of increasing NaCl concentration. Disaccharides are detected by the absorbance at 232 nm due to the presence of the double bond resulting from the action of the lyases. UV detection has a detection limit of ~100 pMoles. The effluent from the UV detector is mixed with NaOH and 2-cyanoacetamide, heated to 130oC to form a fluorescent adduct and then detected fluorometrically. The limit of detection of the fluorscence detection is ~5 pMoles. Control analysis consists of a mixture of known glycosaminoglycan-derived disaccharides. Fractions may be collected for an additional charge. Scintillation counting is also available. The data presented to the investigator consists of the HPLC data trace and a peak table including peak retention times and areas. Peaks co-migrating with known standards will be so marked. Powerpoint formatted data are available for an additional charge.

Pro-007. Profile of Glycosaminoglycan-derived Disaccharides by Ion Pair RP-HPLC with UV and Fluorescent Detection

The starting material for this procedure is typically a mixture of disaccharides obtaining by the action of lyases. This method is prefered for chondroitin- and dermatan-derived disaccharides. Disaccharides are adsorbed to the C18 column by virtue of their ionic interactions with the hydrophobic ion pair reagent tetrabutylammonium hydrogen sulfate. Elution is effected with a gradient of increasing acetonitrile concentration. Disaccharides are detected by the absorbance at 232 nm due to the presence of the double bond resulting from the action of the lyases. UV detection has a detection limit of ~100 pMoles. The effluent from the UV detector is mixed with NaOH and 2-cyanoacetamide, heated to 130oC to form a fluorescent adduct and then detected fluorometrically. The limit of detection of the fluorscence detection is ~5 pMoles. Control analysis consists of a mixture of known glycosaminoglycan-derived disaccharides. Fractions may be collected for an additional charge. Scintillation counting is also available. The data presented to the investigator consists of the HPLC data trace and a peak table including peak retention times and areas. Peaks co-migrating with known standards will be so marked. Powerpoint formatted data are available for an additional charge.

>> more items