To identify novelbinding partners of MGL on Jurkat T-cells, we developed a protocolwhere we used Fc-coupled MGL as a bait in pull-down assays (Figure A)
MGL-Fc is a chimericmolecule formed by the extracellular domains of MGL fused to the human immunoglobulin G1 Fc tail , allowing bindingto magnetic Protein G beads
Because the binding to the CRD of MGLis calcium-dependent, captured proteins were eluted using EDTA andsubsequently analyzed by SDS-PAGE and processed for mass-spectrometry-basedprotein identification
CD43 and CD45 are hitherto the only describedMGL-binding proteins on Jurkat cells
Therefore, to test the effectiveness of our workflow, we first determinedthe capturing of CD43 by Western blot
This clearly showed that CD43was captured by MGL-Fc and could be eluted with EDTA (Figure B)
Likewise, the additionof EDTA during the pull-down assay prevented the binding of CD43 to MGL (Figure B)