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Na, H., Laver, J.D., Jeon, J., Singh, F., Ancevicius, K., Fan, Y., Cao, W.X., Nie, K., Yang, Z., Luo, H., Wang, M., Rissland, O., Westwood, J.T., Kim, P.M., Smibert, C.A., Lipshitz, H.D., Sidhu, S.S. (2016). A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.  RNA 22(4): 636--655.
FlyBase ID
FBrf0231323
Publication Type
Research paper
Abstract
Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.
PubMed ID
PubMed Central ID
PMC4793217 (PMC) (EuropePMC)
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Secondary IDs
    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    RNA
    Title
    RNA (New York, N.Y.)
    Publication Year
    1995-
    ISBN/ISSN
    1355-8382
    Data From Reference