Sialoglycoconjugate analysis
The Sialic Acid Fluorescence Labeling Kit is intended for quantitative and highly sensitive sialoglycoconjugate analysis. Neu5Ac, NeuGc, KDN and their O-acetyl derivatives can each be analyzed by this method.
Sialic acids are one of the most important constituents of glycoconjugates in microorganisms and a wide range of higher order animals. Many sialic acids are bound to sugar chains through alpha-ketoside linkages such as alpha-2,3; alpha-2,6; and alpha-2,8. They are heavily involved in cell-to-cell recognition, blood coagulation, fertilization and other biological events.
The Sialic Acid Fluorescence Labeling Kit is intended for quantitative and highly sensitive sialoglycoconjugate analysis. Neu5Ac, NeuGc, KDN and their O-acetyl derivatives can each be analyzed by this method.
Sialic acids are one of the most important constituents of glycoconjugates in microorganisms and a wide range of higher order animals. Many sialic acids are bound to sugar chains through alpha-ketoside linkages such as alpha-2,3; alpha-2,6; and alpha-2,8. They are heavily involved in cell-to-cell recognition, blood coagulation, fertilization and other biological events.
Conventional methods for sialoglycoconjugate analysis, such as the resorcinol assay and thiobarbituric acid test, involve several complex steps. However, the HPLC-based sialic acid fluorescence labeling kit using 1,2-diamino-4,5-methyleneoxybenzene (DMB) provides a relatively simple and highly sensitive quantitative analysis method. In this method, free sialic acids are analyzed by reverse phase HPLC (PALPAK Type R) after labeling with DMB. Neu5Ac, NeuGc, KDN as well as their O-acetyl derivatives can be individually analyzed through this method, with the detection limit being <57 fmol. Combining DMB labeling with pyridylamino (PA) derivatization results in even higher sensitivity sialoglycoconjugate analysis.
More Information
Applications
- Quantitative and highly sensitive sialoglycoconjugate analysis
- Sialic acid HPLC
Components
| DMB Solution | 2 x 1 ml |
| Coupling Solution* | 2 x 5 ml |
| Neu5Ac (100 µl) | 500 µl |
* Contains 2-mercaptoethanol, sodium hydrosulfite, and acetic acid
Protocol
- Place samples containing free sialic acids (5 pmol–5 nmol) into screw-cap 1.5 ml tubes.
NOTE: The sample volume must be kept under 50 µl. Since acidic conditions are required for the following reaction, the pH must be neutral or acidic. - Prepare a mixed solution with a ratio of Reagent 1:Reagent 2:H2O of 1:5:4. Add 200 µl of the mixed solution to the samples. Mix tubes and allow reactions to proceed for 2.5 hours at 50°C, protected from light.
NOTE: The mixed solution is stable at least for one week at 4°C. - Cool tubes on ice to terminate reactions.
- Inject 10 µl of each reaction mixture into an HPLC column. The quantity of sialic acid within the sample can be calculated from a calibration curve based on the peak height obtained with the defined sialic acid standard.
Storage
Store at –20°C. Protect DMB Solution from light.
Product citations
Hara, S. et al., Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high performance liquid chromatography. Anal. Biochem. 179,162–166 (1989).
Klein, A. et al., New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones. Glycobiology 7, 421–432 (1997).
Nakamura, M. et al., 1, 2-Diamino-4, 5-methylenedioxybenzene as a Highly Sensitive Fluorogenic Reagent for α-Keto Acids. Chem. Pharm. Bull. 35, 687–692 (1987).
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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