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Polyclonal Antibody Development Against Purified CC-NBS-LRR like Protein Fragment from Mature Lageneria siceraria Seeds and Immunolocalization

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Abstract

CC-NBS-LRR (CNL) plant proteins are related with highly conserved family of disease resistance protein distinguished by a coiled-coil domain, which plays an important role in innate immunity. The present study reports the purification and identification of CNL like protein fragment (CNL-LPF) by two step chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/MS), respectively. Furthermore, current study also illustrated the development of polyclonal antibody against purified CNL-LPF, which was used for immunolocalization of CNL-LPF in cytoplasm of cotyledon, using Fluorescence microscopy and Transmission electron microscopy. Lastly, present study also demonstrates in vitro oligomerization of purified CNL-LPF with multiple bands on 4–10 % gradient native-PAGE; each band representing a small fraction of each oligomer population as evident by immunoblots. In conclusion, the current study deals with the purification and polyclonal antibody development against CNL-LPF.

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Abbreviations

CNL:

CC-NBS-LRR protein

CNL-LPF:

CNL like protein fragment

PAbs:

Polyclonal antibody

ELISA:

Enzyme-linked immunosorbent assay

FM:

Fluorescence microscopy

TEM:

Transmission electron microscopy

PM:

Plasma membrane

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Acknowledgments

This work is financially supported by Department of Science and Technology (DST), India) under the Women Scientists Scheme (WOS-A). Neha Kumari thanks DST for providing fellowship. We gratefully acknowledge Dr Radhey Shyam Sharma for fruitful discussion and Dr Kalpana Luthra for allowing us to use her laboratory facility during antibody development.

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Authors and Affiliations

  1. Department of Biophysics, All India Institute of Medical Sciences, New Delhi, 110029, India

    Neha Kumari & Savita Yadav

  2. Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, 121001, India

    Rajesh Kumar

  3. Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, 110029, India

    Rajesh Kumar

  4. Department of Environmental Sciences, University of Delhi, Delhi, 110007, India

    Vandana Mishra

Authors
  1. Neha Kumari
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  2. Rajesh Kumar
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  4. Savita Yadav
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Correspondence to Savita Yadav.

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The authors declare that they have no conflicts of interest.

Ethical Approval

This article contain polyclonal antibody development on New Zealand white rabbits. This study was permitted by the AIIMS animal ethics committee (733/IAEC/13).

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Kumari, N., Kumar, R., Mishra, V. et al. Polyclonal Antibody Development Against Purified CC-NBS-LRR like Protein Fragment from Mature Lageneria siceraria Seeds and Immunolocalization. Protein J 35, 379–390 (2016). https://doi.org/10.1007/s10930-016-9683-9

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  • DOI: https://doi.org/10.1007/s10930-016-9683-9

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