DMB Sialic Acid Labeling Kit
The LudgerTag DMB Sialic Acid Labeling Kit has been developed for the qualitative analysis of sialic acids.
The kit contains reagents for the release of sialic acids from glycoproteins. Released sialic acids are conjugated with DMB dye by an amination-cyclisation reaction.
The kit contains reagents and materials for the release and labeling of either 22 (LT-KDMB-A1) or 96 samples (LT-KDMB-96). The kit also includes the sialic acid reference panel, and the N-acetyl neuraminic acid and N-glycolyl neuraminic acid standards.
LT-KDMB-A1 – 22 samples
LT-KDMB-96 – 96 samples
The DMB sialic acid labeling kit contains two sets of the labeling reagents and a sialic acid reference panel:
– NeuAc and NeuGc quantitative standards
– Sialic Acid Reference Panel
Release and Labeling Reagents
– DMB Dye (1,2-diamino-4,5-methylenedioxybenzene.2HCl)
– Acetic Acid
– Mercaptoethanol in Acetic acid
– Sodium Dithionite (Reductant)
Sialic acids are important for the stability and 3D conformation of therapeutic glycoproteins. They have been shown to affect the serum half-life of glycoproteins and to have an affect on anti-inflammatory and immune response by patients by these biological therapeutics. For these reasons it is imperative to monitor both the level and type of sialic acids found on these therapeutic glycoproteins.
The LudgerTag DMB kit provides all that is required to release sialic acids from glycoproteins and label them with DMB. The kit contains reagents and materials for either 22 or 96 glycoprotein samples (50-100 µg of glycoprotein per sample). Each labeling should work with approximately 10 pmol up to 2.5 nmol sialic acids per sample.
Included in the kit are a sialic acid reference panel (containing Neu5Ac; Neu5Gc; Neu5,7Ac2; Neu5,Gc9Ac; Neu5,9Ac2; Neu5,7,(8),9Ac3Gc) and both NeuAc and NeuGc quantitative standards. These three standards can be labelled with DMB alongside the released sialic acid samples. Preparation of serial dilutions of the NeuAc and NeuGc quantitative standards enables quantitative analysis. Information on how to do this is explained in the LudgerTag DMB kit guide. The NeuAc(NANA) and NeuGc(NGNA) quantitative standards are also available to purchase separately if required.
For positive controls, we recommend using our fetuin glycoprotein which contains both NeuAc and NeuGc sialic acids and/or a quantitative glycopeptide (BQ-GPEP-A2G2S2-10U) as positive controls. Water can be used as a negative control.
Release of Sialic Acids The release of sialic acids from the glycoprotein occurs by a mild acid hydrolysis. Samples and controls are incubated with 2M acetic acid at 80°C for 2 hours.
Sialic Acid Labeling Reaction The released sialic acids are fluorescently labelled with 1,2-diamino-4,5-methylenedioxybenzene-2HCl (DMB). The acid hydrolysed samples and process controls plus Ludger sialic acid standards: Neu5Ac, Neu5Gc, sialic acid reference panel, and Neu5,9Ac2 if using, are incubated with DMB at 50°C for 3 hours.
Analysis via HPLC or UHPLC A standard curve is calculated using the Neu5Ac and Neu5Gc standards. The sialic acid reference panel and Neu5,9Ac2 are used for sialic acid identification. Analysis can be performed using the LudgerSep R1x150mm column for HPLC or via UHPLC using a LudgerSep uR2x100mm column.
Sialic Acid Information Guides
Sialic Acid Labeling Application Notes
DMB Labeling References
Thomson RI, Gardner RA, Strohfeldt K, Fernandes DL, Stafford GP, Spencer DIR, Osborn HMI. Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content. Anal Chem. Jun 20;89(12):6455-6462.( 2017)