Ingrid 't Hart

52 Chapter 2 2 Bio�n-conjugated ganglioside GM1 polyclonal an�body (2 µg/mL; Bioss, bs-2367R- Bio�n) in TSM binding buffer was incubated for 1 h followed by washing as described above. Next the subarray was incubated with Streptavidin-AlexaFluor635 (5 µg/mL) for 1 h followed by washing. Using the same buffers as above, recombinant human Siglec-7 comp (a gi� from Dr. R.L. Schnaar, Johns Hopkins University School of Medicine, Bal�more, MD, USA) was assayed at 50 µg/mL premixed with 6x-His Tag monoclonal an�body-AlexaFluor647 (5 µg/mL; ThermoFisher Scien�fic MA1-135-A647) with an incuba�on for 2 h. All incuba�on and wash steps were performed at RT. Washed arrays were dried by centrifuga�on and immediately scanned for fluorescence on a GenePix 4000 Bmicroarray scanner (Molecular Devices) using a detec�on gain adjusted to avoid satura�on of the signal. The data were processed with GenePix Pro 7 so�ware and further analyzed using our home wri�en Microso� Excel macro. The lowest and highest value of the 6 replicates were excluded, a�er which the mean fluorescence intensi�es (corrected for mean background) and standard devia�ons (SD) were calculated (n=4). Data were fi�ed using Prism so�ware (GraphPad So�ware, Inc). The lowest concentra�on required for good responsiveness in the op�mum dynamic range was selected for all proteins examined. Results and discussion prin�ng controls The prin�ng of the synthe�c compounds was validated by the plant lec�ns MAL II, SBA and WGA and a GM1 an�body. MAL-II binds the terminal trisaccharide sequence Neu5Ac(α2-3)Gal(β1–4)GlcNAc/ Glc. 45 Compounds 17 and 20 (Neu5Ac(α2-8)-Neu5Ac(α2-3)Gal(β1–4)Glc) have three terminal intact sugars, and as expected binds to MAL II. Compounds 5b and 6b , with the Neu5Ac(α2-3)-Gal-β1,3-GalNAc epitope at the terminal end, are not recognized by MAL II. Similarly, GT1b ( 21 ) with the same terminal epitope as 5b and 6b also did not show binding to MAL II. SBA preferen�ally binds GalNAc, and also recognizes Gal residues although at much lower affinity. 46 Binding to SBA was observed for compounds with either a GalNAc or Gal at the terminal residue: 18 (GM2; with GalNAc at the terminal residue) and 4b (Gb5; with Gal at terminal residue). As expected sialylated compounds 5b and 6b (sialylated Gb5; no Gal at the terminal residue) didn’t show any binding. Also compound 19 (GM1a; with Gal at the terminal residue) didn’t show any binding, due to the inhibi�on effect of Neu5Ac. WGA preferen�ally binds GlcNAc moie�es, and also interacts with some glycoproteins via terminal sialic acid residues. Indeed the terminal sialylated compound 21 (GT1b) and sialylated Gb5 ( 5b and 6b ) showed binding, while the non-terminal sialylated compounds 18 (GM2), 19 (GM1a) and 4b (Gb5) did not bind. Compound 20 (GD3; with terminal α2,8-Neu5Ac-a2,3-Neu5Ac) also did not bind to WGA, apparently WGA does not recognize this sialylated epitope. As expected only GM1a ( 19 ) showed binding to the GM1 an�body.