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Production and Characterization of Polyclonal and Monoclonal Abs Against the RNA-Binding Protein QKI

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Abstract

RNA-binding protein QKI, a member of the Signal Transduction and Activation of RNA family, is found to be essential in the blood vessel development and postnatal myelination in central nervous system (Woo et al., Oncogene 28:1176–1186, 2009; Lu et al., Nucleic Acids Res 31(15):4616–4624, 2003; Bohnsack et al., Genesis 44(2):93–104, 2006). However, its wide expression pattern suggests other fundamental roles in vivo (Kondo et al., Mamm Genome 10(7):662–669, 1999). To facilitate the understanding of QKI function in various systems, we prepared the polyclonal and monoclonal antibodies against QKI. To obtain the antigen, recombinant His-tagged QKI was expressed in Escherichia coli and highly purified by Ni2+-chelated column combined with hydrophobic and ion exchange methods. Following three types of immunizations with different adjuvants, including Freund’s, PAGE gel, and nitrocellulose membrane, only the antiserum produced with Freund’s adjuvant is effective for Western blot detection. Several McAb clones are able to recognize both endogenous and over-expressed QKI with high affinity in Western blot and immunofluorescence. The specificity of Ab was validated as weakening, and no specific signals were observed in cells with QKI knocking down. Immunohistochemistry analysis further showed positive staining of QKI in kidney where QKI mRNA was abundantly expressed, ensuring the wide applications of the QKI Abs in the ongoing mechanistic studies.

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Acknowledgements

This work was supported by National Science Foundation of China (NSF: 30470387; NSF: 30570396; NSF: C030200303) and National Key Basic Research and Development Program 2009CB521706.

Conflict of Interest Statement

All authors declare that there are no conflicts of interest.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, the State Key Laboratory of Cancer Biology, The Fourth Military Medical University, No.17 Changlexi Road, 710032, Xi’an, People’s Republic of China

    Jie Zhang, Bo Huang, Fang Yu, Mengying Wei, Guodong Yang, Haiyan Fu, Liang Jin, Liyuan Bai & Zifan Lu

  2. Division of General Surgery, Tangdu Hospital, The Fourth Military Medical University, No.1 Xinshi Road, 710038, Xi’an, People’s Republic of China

    Xianli He

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  1. Jie Zhang
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  2. Bo Huang
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Correspondence to Xianli He or Zifan Lu.

Additional information

Jie Zhang, Bo Huang, and Fang Yu contributed equally to this work and should be considered as co-first authors.

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Zhang, J., Huang, B., Yu, F. et al. Production and Characterization of Polyclonal and Monoclonal Abs Against the RNA-Binding Protein QKI. Appl Biochem Biotechnol 164, 283–293 (2011). https://doi.org/10.1007/s12010-010-9134-6

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  • DOI: https://doi.org/10.1007/s12010-010-9134-6

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