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Validation of Recombinant Antibodies Against Human Transcription Factors
Author(s) -
Griffin Carly,
Miersch Shane,
Marcon Edyta,
Banerjee Sunandan,
Wells Jim,
Hornsby Michael,
Kossiakoff Anthony,
Koide Shohei,
Paduch Marcin,
Sidhu Sachdev,
Moffat Jason
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.571.13
Subject(s) - chromatin immunoprecipitation , computational biology , recombinant dna , transcription factor , immunoprecipitation , biology , microbiology and biotechnology , cell culture , gene expression , promoter , gene , genetics
The Recombinant Antibody Network (RAN) is a pilot project involving 3 integrated automation centres to create renewable, open‐source, high‐quality binding reagents. The main aims of RAN are to: generate rAbs (recombinant antibodies) directed to the ~1500 human transcription factors (TFs) against ~3000 domain targets using high‐throughput selection methods; validate and produce 蠅2 rAbs per target for high performance in applications such as immunofluorescence (IF), western blot, and chromatin‐immunoprecipitation; distribute validation data to our network and the scientific community through a dedicated web portal; and continually improve the efficiency, speed, cost and success. The RAN pipeline progressively identifies clones that bind target domains with high affinity and specificity. Our rAbs should be able to bind and IP full‐length cellular TFs from complex mixtures, and demonstrate specificity between family members. Additional validation steps include over‐expression of the target, knock‐down of the target by RNAi, and altered growth conditions. A key step in the validation process is high‐content screening against a panel of 6 Human cell lines to monitor cellular localization. The rAbs that show nuclear localization by IF are tested by immunoprecipitation‐mass spectrometry for their ability to pull‐down endogenous target. To date, over 1200 rAbs against >250 targets have been monitored by IF, with approximately 25% showing nuclear localization patterns.