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- Boström, ToveKTH,Proteinteknologi (author)
Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry
- Article/chapterEnglish2014
Publisher, publication year, extent ...
- 2014-09-25
- American Chemical Society (ACS),2014
- printrdacarrier
Numbers
- LIBRIS-ID:oai:DiVA.org:kth-154501
- https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-154501URI
- https://doi.org/10.1021/pr500691aDOI
- http://kipublications.ki.se/Default.aspx?queryparsed=id:129888200URI
Supplementary language notes
- Language:English
- Summary in:English
Part of subdatabase
- SwePubSwePub
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Notes
- QC 20141106
- For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.
Subject headings and genre
- Bioteknologi
- Biotechnology
Added entries (persons, corporate bodies, meetings, titles ...)
- Johansson, Henrik J.Karolinska Institutet (author)
- Lehtiö, JanneKarolinska Institutet (author)
- Uhlén, MathiasKTH,Proteomik och nanobioteknologi(Swepub:kth)u1dulvmw (author)
- Hober, SophiaKTH,Proteinteknologi(Swepub:kth)u11qqzc1 (author)
- KTHProteinteknologi (creator_code:org_t)
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- In:Journal of Proteome Research: American Chemical Society (ACS)13:10, s. 4424-44351535-38931535-3907
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