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Preparation and Affinity-Purification of Supervillin Isoform 4 (SV4) Specific Polyclonal Antibodies

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Abstract

Human Supervillin isoform 4 (SV4), a bigger splicing isoform of Supervillin, contains extra coding exons 3, 4 and 5 (E345), compared to Supervillin isoform 1. Although previous studies have shown that SV4 associated with membrane and cytoskeleton, regulated cell migration and cell survival, its functions are still largely unknown. To broaden our understanding, SV4 specific antibody is important for further study in signaling pathway. The His-SV4 (E345) and GST-SV4 (E345) fusion proteins, which contained SV4 specific domain E345, were purified from bacteria. The His-SV4 (E345) proteins were injected in rabbits as immunogen to produce anti-SV4 serum, and SV4 antibodies were purified by GST-SV4 (E345) proteins cross-linked to affinity resins. SV4 antibodies exclusively recognized SV4 protein both in vitro and in vivo through multi-step testing by ELISA, western blot, immunoprecipitation, and immunofluorescence. Taken together, our data demonstrate a novel SV4-specific polyclonal antibody which will provide a useful tool for further characterization of SV4 function.

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Abbreviations

SV:

Supervillin

SV1:

Supervillin isoform 1

SV2:

Supervillin isoform 2

SV4:

Supervillin isoform 4

E345:

Exons 3, 4 and 5

SV4 (E345):

Supervillin isoform 4 specific domain of exons 3, 4 and 5

ELISA:

Enzyme linked immunosorbent assay

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Acknowledgments

We thank the members for technical assistance in Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences. This research was partly supported by the National Natural Science Foundation of China (Nos. 31571433 and 31501171), Anhui Provincial Natural Science Foundation (Nos. 1508085SMC214 and 1608085MH180) and Visiting Professorships Foundation of Hefei Institutes of Physical Science (No. Y3BZOH3058).

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Authors and Affiliations

  1. Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, No. 350, Shushan Hu Road, Hefei, 230031, Anhui, People’s Republic of China

    Xueran Chen, Hao Li, Hongzhi Wang, Chaozhao Liang & Zhiyou Fang

  2. Cancer Hospital, Hefei Institutes of Physical Science, Chinese Academy of Sciences, No. 350, Shushan Hu Road, Hefei, 230031, Anhui, People’s Republic of China

    Hongzhi Wang, Haoran Yang, Fang Ye, Chaozhao Liang & Zhiyou Fang

  3. Department of Urology, The First Affiliated Hospital of Anhui Medical University and Institute of Urology, Anhui Medical University, No. 218, Jixi Road, Hefei, 230022, Anhui, People’s Republic of China

    Chaozhao Liang

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  1. Xueran Chen
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  2. Hao Li
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  3. Hongzhi Wang
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  4. Haoran Yang
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  5. Fang Ye
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  7. Zhiyou Fang
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Correspondence to Zhiyou Fang.

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The authors of this paper declare that they do not have any conflict of interest. And the SV4 antibodies were used for academic research but not for commercial field.

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Chen, X., Li, H., Wang, H. et al. Preparation and Affinity-Purification of Supervillin Isoform 4 (SV4) Specific Polyclonal Antibodies. Protein J 35, 107–114 (2016). https://doi.org/10.1007/s10930-016-9658-x

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  • DOI: https://doi.org/10.1007/s10930-016-9658-x

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