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

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...

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Autores principales: Mesa, Rosana A., Roberson, Elisha D.O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: F1000 Research Limited 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194342/
https://www.ncbi.nlm.nih.gov/pubmed/32399180
http://dx.doi.org/10.12688/f1000research.19803.2
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author Mesa, Rosana A.
Roberson, Elisha D.O.
author_facet Mesa, Rosana A.
Roberson, Elisha D.O.
author_sort Mesa, Rosana A.
collection PubMed
description 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.
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spelling pubmed-71943422020-05-11 Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot Mesa, Rosana A. Roberson, Elisha D.O. F1000Res Antibody Validation Article 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. F1000 Research Limited 2020-01-10 /pmc/articles/PMC7194342/ /pubmed/32399180 http://dx.doi.org/10.12688/f1000research.19803.2 Text en Copyright: © 2020 Mesa RA and Roberson EDO http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Antibody Validation Article
Mesa, Rosana A.
Roberson, Elisha D.O.
Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot
title Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot
title_full Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot
title_fullStr Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot
title_full_unstemmed Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot
title_short Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot
title_sort validation of a commercial antibody to detect endogenous human nicastrin by immunoblot
topic Antibody Validation Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194342/
https://www.ncbi.nlm.nih.gov/pubmed/32399180
http://dx.doi.org/10.12688/f1000research.19803.2
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