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. 2018 Jul 17;28(10):786–801. doi: 10.1093/glycob/cwy057

Fig. 3.

Fig. 3.

Siglec-8-COMP affinity purification of Siglec-8 ligands from human trachea. Siglec-8 ligands extracted from human trachea were resolved by Sephacryl S-500 size-exclusion chromatography (Figure 2) and fractions corresponding to S8-1M, S8-600K and S8-250K separately combined for affinity purification. Pooled fractions of each molecular size were loaded separately onto 1-mL nickel Sepharose affinity columns carrying 6-His-tagged pentameric Siglec-8-COMP. After loading and washing each column, bound Siglec-8 ligands were eluted with high salt. Equal aliquots of washes and eluates were subjected to 1.5% acrylamide, 2% agarose composite gel electrophoresis, blotted to PVDF membranes and probed with Siglec-8-Fc. Lanes: (1) pooled Sephacryl S-500 fraction; (2) affinity column flow-through; (3) wash 1; (4) wash 2; (5) elution 1; (6) elution 2; (7) elution 3. HiMark-prestained molecular weight standards are shown at the left (Stds).