PMID: PMC6399673-1-1

 

    Legend: Gene, Sites

Title : Workflow ofthe MGL Pull-Down Assay

Abstract :
  1. 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)
  2. 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
  3. 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
  4. CD43 and CD45 are hitherto the only describedMGL-binding proteins on Jurkat cells
  5. Therefore, to test the effectiveness of our workflow, we first determinedthe capturing of CD43 by Western blot
  6. This clearly showed that CD43was captured by MGL-Fc and could be eluted with EDTA (Figure B)
  7. Likewise, the additionof EDTA during the pull-down assay prevented the binding of CD43 to MGL (Figure B)
Output (sent_index, trigger, protein, sugar, site):
Output(Part-Of) (sent_index, protein, site):
  • 2. MGL, domains
  • 2. immunoglobulin G1, tail
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX