Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array

Abstract Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. Here, we use microwestern array to rapidly evaluate suitable conditions for quantitative western blotting, with up to 192 antibody/dilution/replicate combinations on a sin...

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Autores principales:	Rick J. Koch, Anne Marie Barrette, Alan D. Stern, Bin Hu, Mehdi Bouhaddou, Evren U. Azeloglu, Ravi Iyengar, Marc R. Birtwistle
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bc7e4c10cd5a4850b9dd736a994f2554
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spelling	oai:doaj.org-article:bc7e4c10cd5a4850b9dd736a994f25542021-12-02T15:08:25ZValidating Antibodies for Quantitative Western Blot Measurements with Microwestern Array10.1038/s41598-018-29436-02045-2322https://doaj.org/article/bc7e4c10cd5a4850b9dd736a994f25542018-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-29436-0https://doaj.org/toc/2045-2322Abstract Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. Here, we use microwestern array to rapidly evaluate suitable conditions for quantitative western blotting, with up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Pilot experiments demonstrate a high proportion of investigated antibodies (17/24) are suitable for quantitative use; however this sample of antibodies is not yet comprehensive across companies, molecular weights, and other important antibody properties, so the ubiquity of this property cannot yet be determined. In some cases microwestern struggled with higher molecular weight membrane proteins, so the technique may not be uniformly applicable to all validation tasks. Linear range for all validated antibodies is at least 8-fold, and up to two orders of magnitude. Phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection. Total antibodies generally required higher working concentrations, but more comprehensive antibody panels are required to better establish whether this trend is general or not. Importantly, we demonstrate that results from microwestern analyses scale to normal “macro” western for a subset of antibodies.Rick J. KochAnne Marie BarretteAlan D. SternBin HuMehdi BouhaddouEvren U. AzelogluRavi IyengarMarc R. BirtwistleNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-10 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rick J. Koch Anne Marie Barrette Alan D. Stern Bin Hu Mehdi Bouhaddou Evren U. Azeloglu Ravi Iyengar Marc R. Birtwistle Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array
description	Abstract Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. Here, we use microwestern array to rapidly evaluate suitable conditions for quantitative western blotting, with up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Pilot experiments demonstrate a high proportion of investigated antibodies (17/24) are suitable for quantitative use; however this sample of antibodies is not yet comprehensive across companies, molecular weights, and other important antibody properties, so the ubiquity of this property cannot yet be determined. In some cases microwestern struggled with higher molecular weight membrane proteins, so the technique may not be uniformly applicable to all validation tasks. Linear range for all validated antibodies is at least 8-fold, and up to two orders of magnitude. Phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection. Total antibodies generally required higher working concentrations, but more comprehensive antibody panels are required to better establish whether this trend is general or not. Importantly, we demonstrate that results from microwestern analyses scale to normal “macro” western for a subset of antibodies.
format	article
author	Rick J. Koch Anne Marie Barrette Alan D. Stern Bin Hu Mehdi Bouhaddou Evren U. Azeloglu Ravi Iyengar Marc R. Birtwistle
author_facet	Rick J. Koch Anne Marie Barrette Alan D. Stern Bin Hu Mehdi Bouhaddou Evren U. Azeloglu Ravi Iyengar Marc R. Birtwistle
author_sort	Rick J. Koch
title	Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array
title_short	Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array
title_full	Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array
title_fullStr	Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array
title_full_unstemmed	Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array
title_sort	validating antibodies for quantitative western blot measurements with microwestern array
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/bc7e4c10cd5a4850b9dd736a994f2554
work_keys_str_mv	AT rickjkoch validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray AT annemariebarrette validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray AT alandstern validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray AT binhu validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray AT mehdibouhaddou validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray AT evrenuazeloglu validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray AT raviiyengar validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray AT marcrbirtwistle validatingantibodiesforquantitativewesternblotmeasurementswithmicrowesternarray
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Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array

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