Lectin Array 95

Introduction The  lectin array uses standard glass slides each spotted with 8 wells of identical lectin arrays. Each lectin, together with the positive controls is arrayed in duplicate. The slides each come with a 8-well removable gasket which allows for the process of 8 samples using one slide. Four slide slides can be nested into a tray, which matches a standard microplate and allows for automated robotic high throughput process of 32 arrays simultaneously. The lectin array provides a powerful new tool for glycosylation determination, drug discovery and biomarker development; all with limited samples volumes required. Product Feature High sensitivity and specificity Low sample volume (10-100 µl per array) Large dynamic range of detection Compatible with most sample types Test 12 samples on each slide Suitable for high-throughput assays Lectin Names AAA, AAL, ABA, ABL, ACG, ACL, AMA, ASA, BanLec, BC2L-A, BC2LCN, BPA, CA, CAA, Calsepa, CGL2, CNL, Con A, CPA, CSA, DBA, Discoidin I, Discoidin II, DSA, ECA, EEL, F17AG, Gal1, Gal1-S, Gal2, Gal3, Gal3C-S, Gal7-S, Gal9, GHA, GNA, GRFT, GS-I, GS-II, HAA, HHA, HMA, IRA, Jacalin, LAL, LBA, LCA, LEA, Lentil, Lotus, LPA, LSL-N, MAA, Malectin, MNA-G, MNA-M, MOA, MPL, NPA, Orysata, PA-IIL, PA-IL, PALa, PHA-E, PHA-L, PHA-P, PNA, PPL, PSA, PSL1a, PTL-1, PTL-2, PWA, RCA-120, RCA-60, RPA, RS-Fuc, SAMB, SBA, SHA, SJA, SNA-I, SNA-II, SSA, STL, TL, UDA, UEA-I, UEA-II, VFA, VRA, VVA, VVA-M, WFA, WGA Application Data/Notes Application 1 - Detection of Glycans on a Purified Protein See Image/s In this application, the Lectin Array 95 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 signals 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 95 were consistent with published literature regarding HRP glycosylation. Application 2 - Profiling of a Serum Sample See Image/s Using the lectin 95 array, we can discover the different glycoprotein profiles of serum samples, cell lysates, or purified glycoproteins. The images above show the profiles of the glycans from different types of samples including human serum, recombinant glycoproteins human HE4 and AFP, mouse TFF2, purified human IgG, and bacterial DE3 cell lysates detected by Biotin labeling and fluorescent dye-streptavidin. Suggested Applications 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 Other Applications 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. Example: Cell lysate from bacteria can be Biotinylated and hybridized 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. Kit Components 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 Other Materials Required 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, KH 2 PO 4  and Na 2 HPO 4  (For label-based method only) Plastic or glass containers, beaker, stir plate and stir bar Pipettors, pipette tips, ddH 2 O and other common lab consumables Protocol Outline Dry the glass slide Block array surfaces Incubate samples (samples need to be biotinylated for the label-based approach) For the the sandwich-based principle, incubate with a detection antibody cocktail For the label-based principle, incubate the labeled-streptavidin. Incubate with Cy3 Equivalent Dye-Streptavidin Disassemble the glass slide Scan with a  gene microarray laser scanner Perform densitometry and analysis Storage/Stability Upon receipt, all components of the Lectin Array 95 kit should be stored at -20 ° C. Once thawed, the glass slide and Cy3 equivalent dye-conjugated Streptavidin should be kept at –20 ° C and all other components may be stored at 4 ° C. The entire kit should be used within 6 months of purchase.