MassIVE MSV000100018

Partial Public PXD071203

CD4+ T cell surface biotinylation using pre-complexed Siglec-10 Fc and Protein A-APEX2

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Description

Follicular CD4+ T cells were enriched from pooled splenocytes in immunized C57BL/6J mice. 1 million cells per well in a 96-well V-bottom plate were incubated with 1X PBS containing 2 ug/mL Siglec-10 Fc (or Siglec-10 R119A Fc) pre-complexed with hemin-containing Protein A-APEX2 (1:2 molar ratio) for 30 min at RT, washed with 1X PBS and incubated with 500 uM biotin-tyramide (Millipore) in 1X PBS for 30 min at RT. Cells were then washed with 1X PBS and incubated with 500 uM H2O2 for 1 min at RT to initiate protein biotinylation. Reaction was quenched by washing cells 3 times with 1X PBS supplemented with 5 mM Trolox (Millipore) and 10 mM sodium ascorbate. Cell pellets were lysed in RIPA lysis buffer with protease inhibitors and underwent automated streptavidin beads enrichment (Pierce PI88817) using KingFisher Duo Prime and on-bead trypsin digestion. 10R: Sig-10 R119A Fc; 10W: Sig-10 WT Fc. [doi:10.25345/C52805C1C] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Siglec-G ; B cells ; APEX ; Cell surface biotinylation ; Proximity labeling ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Matthew Macauley, University of Alberta, Canada
Olivier Julien, University of Alberta, Canada
Submitting User: julienlab
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