• Preparations
  • Modification
  • Profile
  • Composition
  • Miscellaneous
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Pre-001: Preparation of mixed N-Glycans.

Releasing N-glycans from either glycoprotein or glycopeptides by using PNase F enzyme and released with C18 and PGC cartridges.

Pre-002: Preparation of Mixed O-Glycans

Releasing O-glycans from either glycoprotein or glycopeptides by doing reductive β-elimination.

Pre-003: Preparation of Mixed Glycolipid-derived Glycans

Releasing glycans from glycolipid by treating with specific endo-glycoceramidase.

Pre-004: Preparation of Total Lipid Extracts

Using Organic solvents to remove total lipids.

Pre-005: Preparation of Cellular Metabolites-TCA Extraction

TCA extracted by using Freon: trictylamine (3:1) from tissue or cell pellets.

Pre-006: Preparation of Glycosaminoglycans

Tissue or Cell digested in Pronase or Protease enzyme and glycosaminoglyca (GAG) released using DEAE column.

Pre-007: Preparation of Glycopeptides

Protein are denatured using Trypsin.

Pre-008: Analysis of Sulfate and Phosphate

Sulfate and Phosphate are released by hydrochloric acid.

Pre-009: Extraction of Semi-Rough or Smooth LPS (Hot Phenol water extraction)

Bacterial cells are washed by PBS and Hot Phenol extraction the LPS are removed. The extracted LPS are treated with DNase/RNase and Proteinase K to remove contaminating nucleic acid and proteins respectively.

Pre-010: Extraction of Rough LPS (Phenol-Chloroform-Petroleum Ether extraction method)

Bacterial cells dried grinded to fine powder and reaction mixture in the ratio of 2:5:8 of 90% phenol: chloroform: petroleum-ether. Rough LPS is precipitated from the phenol layer.

Pre-011: Capsular polysaccharide extraction

CPS are isolated from supernatant by cold absolute ethanol precipitation.

Pre-012: Bacterial Lipid Extraction

Bacterial lipids are extracted from pelleted cells using Bligh-Dyers extraction protocol.

Pre-013: Sugar nucleotide extraction

Lyse cells are dissolved in 70% EtOH and centrifuge to isolate sugar nucleotide.

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Mod 001:Alditol Acetate derivative for GC-MS (AA)

Dry glycan samples are hydrolyzed, reduced by Sodium borohydride than neutralize and acetylated than suspended in dichloromethane and ready for injection on GC-MS.

Mod 002:Trimethyl Silyl derivative for GC-MS (TMS)

Materials for this procedure are methanolysed, re-N-acetylated, treated with Tri-Sil extracted with hexane and ready for injection on GC-MS.

Mod 003a: Per-O-Methylation of glycans (PM)

Sample is dissolved in dry DMSO and Sodium hydroxide powder mixed with DMSO to make slurry and methyl iodide added to permethylated oligosaccharide are analyzed by MALDI mass spectrometry or as PMAA derivative for linkage analysis.

Mod 003b:Partially methylated Alditol Acetate (PMAA)

Partially Methylated Alditol Acetate derivative is done after permethylation of glycans as described in Mod 003a than hydrolyzed, reduced and acetated describe in Mod-001. The linkage analysis is performed on a Gas-Chromatography Mass spectrometry instrument.

Mod 004: Fatty acid methyl ester (FAME)

Dried samples are methanolysed, half-saturated NaCl is added to the samples and fatty acid methyl ester is extracted using chloroform. Fatty acid samples are dissolved in hexane and injected on GC-MS.

Mod 005: Mild acid treatment for releasing Sialic acid

Materials are hydrolyzed in Acetic Acid extracted with 10K micron filter and dried for further analysis.

Mod 006: De-O-Acetylation of glycans

De-O-Acetylation of Glycans treated with base 50mM of Sodium.

Mod-007: Enzymatic degradation of Chondroitin Sulfate (GAG-CS)

An Individual enzyme or a cocktail mixture of Chondroitinase ABC is added for polysaccharide digestion.

Mod-008: Enzymatic degradation of Heparin Sulfate (GAG-HS)

An Individual enzyme or a cocktail mixture of Heparinase I, II, III is added for polysaccharide digestion.

Mod-009: Isotopic Aniline tagging for GAG disaccharide analysis by mass spectrometry (GRIL-Glycan Reductive Isotope Labeling)

Reductive isotope labeling was performed by adding 12C6 aniline or 13C6 aniline to freshly prepared buffer after enzymatic digestion as describe in Mod-007 or Mod-008.

Mod-010: 2AB labeling of glycans

2-Aminobenzamide (2-AB) fluorescence tagged to reducing end of N-glycans and analyzed using Pro-003 method.

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Pro-001: HPAEC-PAD Profile of N-Glycans

Prepared samples as described in Pre-001 and analyzed by HPEAC-PAD.
Example

Pro-002: O-Glycan profiling

Prepared samples as described in Pre-002 and analyzed by HPEAC-PAD.
Example

Pro-003: 2AB-labelled glycan profiling

Prepared samples as described in Mod-010 and analyzed by HPAEC-FL.
Example

Pro-004: Profile of Glycosaminoglycan-derived Disaccharides by Ion Pair RP-HPLC with UV and Fluorescent Detection

Prepared samples as described in Mod-007 and analyzed by HPLC-FL with Post Column.
Example

Pro-005: Profile of Glycosaminoglycan-derived Disaccharides by Anion Exchange HPLC with UV and Fluorescent Detection

Prepared samples as described in Mod-008 and analyzed by HPLC-FL with Post Column.
Example

Pro-006: Single HPLC run

(No sample preparation based on hourly charge) Sample and column conditions are variable according to requirements. This is a limited service for more information contact core Director.
Example

Pro-007: MALDI-TOF profiling of glycans

Prepared samples as described in Mod-003a and analyzed by MALDI.
Example

Pro-008: Cellular metabolite analysis

Prepared samples as described in Pre-005 and analyzed by HPEAC-UV.
Example

Pro-009: GRIL LCQ-MS for GAG disacchar Analysis

Prepared samples as described in Mod-007 or Mod-008 are tagged with aniline as described in Mod-009 and analyzed by LCQ-MS.

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Com-001: Monosaccharide analysis by HPAEC-PAD

The starting material for this procedure is typically glycoprotein, glycolipid or glycan are hydrolyzed by TFA and analyzed by HPAEC-PAD.
Example

Com-002: Uronic acid analysis by HPAEC-PAD

The starting material for this procedure is typically any proteoglycan, plant polysaccharide or bacteria-derived glycan are hydrolyzed by TFA and analyzed by HPAEC-PAD.
Example

Com-003: Sialic acid analysis by HPAEC-PAD

The starting material hydrolyzed by Acetic Acid and analyzed by HPAEC-PAD.
Example

Com-004: Analysis of Anions by Ion Chromatography

The starting material are prepared as described in Pre-008 and analyzed by HPEAC-CD.
Example

Com-005: Reverse phase HPLC of DMB-Sialic acid and determination by Fluorescence detector

Prepared samples as described in Mod-005 and derivatized with DMB and analyzed by HPLC-FL.
Example

Com-006: GC-MS of TMS derivative

Prepared samples described in Mod-002 are Trimethylsilyl derivatives and analyzed by Agilent GC 7820A-MS 5975.
Example

Com-007: GC-MS of AA derivative

Prepared samples as described in Mod-001 and analyzed by Agilent GC 7820A-MS 5975.
Example

Com-008: GC-MS of PMAA derivative (linkage analysis)

Prepared samples as described in Mod-003 treated with Ciucanu-Kerek or modified method analyzed by Agilent GC 7820A-MS 5975.
Example

Com-009: GC-MS of Fatty acids

Prepared samples as described in Mod-004 and analyzed by GC 7820A-MS 5975.
Example

Com-010: LCQ-MS Single runs no sample prep (Per hour)

Suitable sample are dissolved organic solvent and run on either positive or negative mode analyzed on LCQ-MS.

Com-011: Glycan analysis by LCQ-MS

Glycan samples are separated on suitable column (reverse phase or amino-bonded column) analyzed on LCQ-MS.

Com-012: Nucleotide sugar analysis by HPAEC-UV

Prepared samples as described in Pre-013 and analyzed by HPEAC-UV.

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Mis-001: Consultation Time (Per Hour)

It is the policy of the GlycoAnalytics Core that each project be given a maximum of 30 minutes of discussion at no charge with the Core Director. Additional time for individualized analysis of the problem, help in selecting the best protocol for the isolation, purification and analysis of glycoconjugates and interpretation of data is available through consultation with the Core Director, depending upon available time.

Mis-002: Additional Analytical Time (Per Hour)

There may be an additional charge for customizing methods or rushed analysis.

Mis-003: Method development based on time and materials used

Glycotechnology Core is always willing to do a method development and discover new strategies for analyzing. A fee will be charge for the establishment of a new method and the subsequent transfer of the method to the customer.

Mis-004: Fraction collection by HPLC (Per Fraction)

GlycoAnalytics Core can collect fractions from HPLC for the customer.

Mis-005: Powerpoint slide preparation.

Many of the instruments in Glycotechnology Core utilize proprietary software that is not commonly found in most research laboratories.

Mis-006: NMR analysis on the glycan samples will be done upon request.

Contact the Core Director for a price quote.

Glycotechnology Core Resource • University of California, San Diego • 9500 Gilman Drive, BRF2, Room 4243, La Jolla, CA 92093-0687