In this application, the RayBio Lectin Array 70 was used to detect specific glycosylations of purified Horseradish Peroxidase (HRP). Lectins BANLEC, BC2L-A, CALSEPA, GNA, HHA, NPA, PA-IIL, and PALa showed strong signals after incubation with 3.3 ug/mL Biotin-HRP followed by detection with streptavidin-fluorescence-dye (Figures A, B and C). The fluorescence signals from BANLEC, BC2L-A, CALSEPA, GNA, HHA, NPA, PA-IIL, and PALa were blocked in a concentration-dependent manner by HRP itself (Figures A and C), indicating that the signals were generated by lectin-HRP binding. These eight lectins are known to exhibit specific binding to mannose, which indicates that HRP contains mannose. After adding increasing amounts of mannose, the signal from BANLEC, BC2L-A, CALSEPA, GNA, HHA, NPA, PA-IIL, and PALa were reduced (Figures A and B).The reduction in signals from increasing concentrations of mannose confirms that HRP protein contains mannose in its glycocalyx. Additionally, the two lectins AAL and RS-FUC (fucose binding specificity) also showed strong interaction with HRP, which indicates the fucosylation of HRP. Overall, the results of the Lectin Array 70 were consistent with published literature regarding HRP glycosylation.

Using the lectin array, we can discover the different glycoprotein profiles of the serum samples, cell lysates, or purified glycoprotein. The images above show the profiles of the glycans from different types of samples including human serum, recombinant glycoproteins human HE4, AFP, mouse TTF, purified human IgG, and bacterial cell lysates OF DH-5α, DE3 detected by Biotin labeling and Fluorescence dye-streptavidin.

• Identify and profile the glycans in their samples
• Determine whether their biomarker of interest has glycan moieties
• Find specific glycan binding ligands in biological samples

Quantitative analysis of lectin-glycoprotein interactions. Example: a concentration series of glycoproteins detected with the lectin array could reveal concentration dependent effects of lectin-glycan binding.

Determine the profile of bacterial cell-surface glycans; cell lysate from bacteria can be biotinylated andhybridized to the lectin array. Analysis of the binding pattern and correlation with the known carbohydrate-binding specificities of the lectins can determine the glycans on the cell membrane.

• Dialysis Vials
• Labeling Reagent
• Labeling Buffer
• Stop Solution
• Lectin Array Glass Slide Assembly
• Sample Diluent
• 20X Wash Buffer I
• 20X Wash Buffer II
• Cy3 equivalent dye-conjugated Streptavidin
• Slide Washer/Dryer
• Adhesive device sealer
• Floating Dialysis Rack
• Manual

• Detection antibodies of interest (For sandwich-based method only)
• Orbital shaker
• Laser scanner for fluorescence detection
• Aluminum foil
• 1.5ml Polypropylene microcentrifuge tubes
• KCl, NaCl, KH₂PO₄ and Na₂HPO₄ (For label-based method only)
• Plastic or glass containers, beaker, stir plate and stir bar
• Pipettors, pipette tips, ddH₂O and other common lab consumables

1. Dry the glass slide
2. Block array surfaces
3. Incubate samples (samples need to be biotinylated for the label-based approach)
4. For the the sandwich-based principle, incubate with a detection antibody cocktail
   For the label-based principle, incubate the labeled-streptavidin.
5. Incubate with Cy3 Equivalent Dye-Streptavidin
6. Disassemble the glass slide
7. Scan with a gene microarray laser scanner
8. Perform densitometry and analysis