Figure 3.

Purification of Siglec-7 Ligands for MS Analysis. Ligands were purified from the crude membrane fraction (2 mg) and the whole cell lysate (6 mg) of K562 using Siglec-7EcFc (125 μg). Ligands were competitively eluted using 50 nM Dex (Dex) and diSia-Dex (diSia) three times and concentrated by ultrafiltration. Each sample and the eluted beads (gel) were heat denatured in sample buffer and separated using SDS-PAGE. The proteins were visualized using silver staining for MS analysis (Silver staining); western blotting was also performed to confirm the molecular weight of the ligands using Siglec-7EcFc (Sig7EcFc). Bands shown as asterisks at 110 kDa were excised from the gel and used for further sample preparation for MS.