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Affinity purification mass spectrometry

ABSTRACT: To identify potential ligands of Siglec-G in the GBM microenvironment, a Siglec-G-Fc pull-down assay followed by mass spectrometry analysis was performed. Briefly, recombinant Siglec-G-Fc (Novoprotein, Cat. No.:C05K) fusion protein containing the extracellular domain of Siglec-G fused to the human IgG1 Fc region was incubated with either glioma tumor tissue lysates derived from tumor-bearing mice or GL261 glioma cell lysates. Human IgG1-Fc (Novoprotein, Cat. No.:C30D) was used as a negative control. The mixtures were incubated at 4 °C with rotation to allow binding between Siglec-G and potential ligands. Immune complexes were captured using Protein A/G agarose beads and extensively washed with lysis buffer to remove non-specifically bound proteins. Bound proteins were subsequently eluted and separated by SDS-PAGE. Gel bands were excised and subjected to in-gel trypsin digestion. The resulting peptides were analyzed by LC-MS. Candidate ligands were identified by comparing proteins enriched in the Siglec-G-Fc samples relative to the IgG1-Fc control. Proteins were further filtered based on peptide abundance and cell surface localization to prioritize potential Siglec-G-binding ligands.

ORGANISM(S): Mus Musculus

SUBMITTER: Yanan Wang

PROVIDER: PXD075899 | iProX | Thu Mar 19 00:00:00 GMT 2026

REPOSITORIES: iProX

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