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Products >  Glycobiology >  Glycobiology_Kits >  Sialic_Acid_Kit

Sialoglycoconjugate Analysis: Sialic Acid Fluorescence Labeling Kit

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.

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Applications

  • Quantitative and highly sensitive sialoglycoconjugate analysis
  • Sialic acid HPLC

Components

DMB Solution2 x 1 mL
Coupling Solution*2 x 5 mL
Neu5Ac (100 µL)500 µL

*Contains 2-mercaptoethanol, sodium hydrosulfite, acetic acid

Protocol

  1. 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.
  2. 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.
  3. Cool tubes on ice to terminate reactions.
  4. 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.

References

  1. Klein, A. et al. (1997) Glycobiology 7:421-432.
  2. Hara, S. et al. (1989) Anal. Biochem. 179:162-166.
  3. Nakamura, M. et al. (1987) Chem. Pharm. Bull. 35:687-692.



 
 
Products
Cat. # Product Contents Size Price Units Select
4400 Sialic Acid Fluorescence Labeling Kit 100 Rxns $810.00
 

 

 
 

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