Immunodetection of P-selectin using an antibody to its C-terminal tag

Methods Mol Biol. 2015:1312:293-9. doi: 10.1007/978-1-4939-2694-7_31.

Abstract

P-selectin is a multi-domain glycoprotein expressed on activated endothelial cells and activated platelets. We previously expressed a recombinant form of P-selectin containing only its N-terminal lectin and EGF domains in CHO-K1 cells and showed that these two domains are sufficient to mediate ligand binding. We have now expressed the same construct in CHO-Lec1 cells that make truncated glycans. The uniform glycosylation in these cells should make it easier to crystallize this protein.

MeSH terms

  • Animals
  • Antibodies / immunology*
  • Blotting, Western
  • CHO Cells
  • Chromatography, Affinity
  • Cricetinae
  • Cricetulus
  • Electrophoresis, Polyacrylamide Gel
  • Epidermal Growth Factor / chemistry
  • Humans
  • Immunoassay / methods*
  • P-Selectin / analysis*
  • P-Selectin / chemistry*
  • P-Selectin / immunology
  • P-Selectin / isolation & purification
  • Protein Structure, Tertiary
  • Silver Staining
  • Solubility

Substances

  • Antibodies
  • P-Selectin
  • Epidermal Growth Factor