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Validation of antibodies for Western blot applications using orthogonal methods
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. (Uhlen lab)ORCID iD: 0000-0002-0017-7987
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-7692-1100
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

There is a great need for standardized validation methods for antibody specificity and selectivity. Here, we describe the use of orthogonal methods in which the specificity of an antibody in a particular application is determined based on correlation of protein abundance across several samples using an antibody-independent method. We show that pair-wise correlation between orthogonal samples can be used to score the specificity of antibodies in a standardized manner using a test panel of human cell lines. Here, we investigated two independent methods for validation of antibodies in Western blot applications, namely transcriptomics and targeted proteomics and we show that the two methods yield similar, but not identical results. The orthogonal methods can also be used to investigate on- and off- target binding for antibodies with multiple bands in the Western blot assay. In conclusion, orthogonal methods for antibody validation provide an attractive strategy for systematic validation of antibodies in a quantitative manner. 

National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-193962OAI: oai:DiVA.org:kth-193962DiVA: diva2:1034916
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-13Bibliographically approved
In thesis
1. Targeted proteomics methods for protein quantification of human cells, tissues and blood
Open this publication in new window or tab >>Targeted proteomics methods for protein quantification of human cells, tissues and blood
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The common concept in this thesis was to adapt and develop quantitative mass spectrometric assays focusing on reagents originating from the Human Protein Atlas project to quantify proteins in human cell lines, tissues and blood. The work is based around stable isotope labeled protein fragment standards that each represent a small part of a human protein-coding gene. This thesis shows how they can be used in various formats to describe the protein landscape and be used to standardize mass spectrometry experiments. The first part of the thesis describes the use of antibodies in combination with heavy stable isotope labeled antigens to establish a semi-automated protocol for protein quantification of complex samples with fast analysis time  (Paper~I). Paper II introduces a semi-automated cloning protocol that can be used to selectively clone variants of recombinant proteins, and highlights the automation process that is necessary for large-scale proteomics endeavors. This paper also describes the technology that was used to clone all protein standards that are used in all of the included papers.

                     

The second part of the thesis includes papers that focus on the generation and application of antibody-free targeted mass spectrometry methods. Here, absolute protein copy numbers were determined across human cell lines and tissues (Paper III) and the protein data was correlated against transcriptomics data. Proteins were quantified to validate antibodies in a novel method that evaluates antibodies based on differential protein expression across multiple cell lines (Paper IV). Finally, a large-scale study was performed to generate targeted proteomics assays (Paper V) based on protein fragments. Here, assay coordinates were mapped for more than 10,000 human protein-coding genes and a subset of peptides was thereafter used to determine absolute protein levels of 49 proteins in human serum.

                     

In conclusion, this thesis describes the development of methods for protein quantification by targeted mass spectrometry and the use of recombinant protein fragment standards as the common denominator.

Place, publisher, year, edition, pages
Stockholm: Kungliga Tekniska högskolan, 2016. 90 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:16
Keyword
proteomics, mass spectrometry, protein quantification, stable isotope standard, parallel reaction monitoring, immuno-enrichment
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-193951 (URN)978-91-7729-153-4 (ISBN)
Public defence
2016-11-11, Gard-aulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (English)
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Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-14Bibliographically approved

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Edfors, FredrikLinderbäck, KlasSivertsson, ÅsaDanielsson, FridaFagerberg, LinnLundberg, EmmaAlm, ToveForsström, BjörnUhlén, Mathias
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