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Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR

In vivo biotinylation using wild-type and mutants of biotin ligases is now widely applied for the study of cellular proteomes. The commercial availability of kits for the highly efficient purification of biotinylated proteins and their excellent compatibility with LC-MS/MS protocols are the main rea...

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Autores principales: Kanayev, Darkhan, Abilmazhenova, Diana, Akhmetollayev, Ilyas, Sekenova, Aliya, Ogay, Vyacheslav, Kulyyassov, Arman
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9862934/
https://www.ncbi.nlm.nih.gov/pubmed/36676054
http://dx.doi.org/10.3390/life13010107
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author Kanayev, Darkhan
Abilmazhenova, Diana
Akhmetollayev, Ilyas
Sekenova, Aliya
Ogay, Vyacheslav
Kulyyassov, Arman
author_facet Kanayev, Darkhan
Abilmazhenova, Diana
Akhmetollayev, Ilyas
Sekenova, Aliya
Ogay, Vyacheslav
Kulyyassov, Arman
author_sort Kanayev, Darkhan
collection PubMed
description In vivo biotinylation using wild-type and mutants of biotin ligases is now widely applied for the study of cellular proteomes. The commercial availability of kits for the highly efficient purification of biotinylated proteins and their excellent compatibility with LC-MS/MS protocols are the main reasons for the choice of biotin ligases. Since they are all enzymes, however, just a very low expression in cells is required for experiments. Therefore, it can be difficult to perform the quantifications of these enzymes in various samples. Traditional methods, such as western blotting, are not always fit for the detection of the expression levels. Therefore, real-time qRT-PCR, a technology that is more sensitive, was used in this study to quantify the expression of BirA fusions. Using this method, we detected high expression levels of BirA fusions in models of interactions of pluripotency transcription factors to carry out their relative quantification. We also found the absence of the competing endogenous proteins SOX2 and OCT4, as well as no cross-reactivity between BAP/BirA and the endogenous biotinylation system in HEK293T cells. Thus, these data indicated that the high level of biotinylation is due to the in vivo interaction of BAP-X and BirA-Y (X,Y = SOX2, OCT4) in the cell rather than their random collision, a big difference in the expression level of BirA fusions across samples or endogenous biotinylation.
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spelling pubmed-98629342023-01-22 Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR Kanayev, Darkhan Abilmazhenova, Diana Akhmetollayev, Ilyas Sekenova, Aliya Ogay, Vyacheslav Kulyyassov, Arman Life (Basel) Communication In vivo biotinylation using wild-type and mutants of biotin ligases is now widely applied for the study of cellular proteomes. The commercial availability of kits for the highly efficient purification of biotinylated proteins and their excellent compatibility with LC-MS/MS protocols are the main reasons for the choice of biotin ligases. Since they are all enzymes, however, just a very low expression in cells is required for experiments. Therefore, it can be difficult to perform the quantifications of these enzymes in various samples. Traditional methods, such as western blotting, are not always fit for the detection of the expression levels. Therefore, real-time qRT-PCR, a technology that is more sensitive, was used in this study to quantify the expression of BirA fusions. Using this method, we detected high expression levels of BirA fusions in models of interactions of pluripotency transcription factors to carry out their relative quantification. We also found the absence of the competing endogenous proteins SOX2 and OCT4, as well as no cross-reactivity between BAP/BirA and the endogenous biotinylation system in HEK293T cells. Thus, these data indicated that the high level of biotinylation is due to the in vivo interaction of BAP-X and BirA-Y (X,Y = SOX2, OCT4) in the cell rather than their random collision, a big difference in the expression level of BirA fusions across samples or endogenous biotinylation. MDPI 2022-12-30 /pmc/articles/PMC9862934/ /pubmed/36676054 http://dx.doi.org/10.3390/life13010107 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Kanayev, Darkhan
Abilmazhenova, Diana
Akhmetollayev, Ilyas
Sekenova, Aliya
Ogay, Vyacheslav
Kulyyassov, Arman
Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR
title Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR
title_full Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR
title_fullStr Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR
title_full_unstemmed Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR
title_short Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR
title_sort detection of recombinant proteins sox2 and oct4 interacting in hek293t cells using real-time quantitative pcr
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9862934/
https://www.ncbi.nlm.nih.gov/pubmed/36676054
http://dx.doi.org/10.3390/life13010107
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