Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot

Elisha D.O. Roberson, Rosana A. Mesa

Research output: Contribution to journalArticlepeer-review

Abstract

Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.

Original languageEnglish
Article number1211
JournalF1000Research
Volume8
DOIs
StatePublished - 2020

Keywords

  • Gamma secretase
  • Human
  • Nicastrin
  • Polyclonal
  • Western blot

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