Advertisement

Sertoli Cell Preparation for Co-immunoprecipitation

  • Maria João Freitas
  • Margarida FardilhaEmail author
Protocol
  • 737 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1748)

Abstract

Most techniques to study protein-protein interactions, gene expression or signal transduction, among others, in Sertoli cells, depend on obtaining a protein extract of such cells. This is accomplished by lysing the Sertoli cells and solubilizing the intracellular proteins. Depending on the purpose of your study, the technique used to lyse and consequently obtain protein extracts from Sertoli cells must be considered. In this chapter, we will focus on how to obtain a protein Sertoli cell extract suitable for co-immunoprecipitation technique.

Keywords

Lysis buffer Protein quantification Co-immunoprecipitation Sertoli cells Western blot 

Notes

Acknowledgments

This work was supported by FEDER funds through the “Programa Operacional Competitividade e Internacionalização (COMPETE 2020)” and by national funds through the FCT (Fundação para a Ciência e Tecnologia), Projects PTDB/BBB-BQB/3804/2014 and PTDC/DTP-DES/6077/2014. We are thankful to the Institute for Biomedicine (iBiMED) (UID/BIM/04501/2013) for supporting this project. iBiMED is supported by the Portuguese Foundation for Science and Technology (FCT), Compete2020, and FEDER fund. This work was also supported by an individual grant from FCT of the Portuguese Ministry of Science and Higher Education to M.J.F. (SFRH/BD/84876/2012).

References

  1. 1.
    Goldring JP (2012) Protein quantification methods to determine protein concentration prior to electrophoresis. Methods Mol Biol 869:29–35. https://doi.org/10.1007/978-1-61779-821-4_3 CrossRefPubMedGoogle Scholar
  2. 2.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85CrossRefPubMedGoogle Scholar
  3. 3.
    Fasman GD (1989) Practical handbook of biochemistry and molecular biology. CRC Press, Boca Raton, FLGoogle Scholar
  4. 4.
    EMBL (2017) Protein purification - extraction and clarification choice of lysis buffer and additives. https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/. Accessed 5 Mar 2017
  5. 5.
    Linke D (2009) Detergents: an overview. Methods Enzymol 463:603–617. https://doi.org/10.1016/s0076-6879(09)63034-2 CrossRefPubMedGoogle Scholar
  6. 6.
    Paasch U, Heidenreich F, Pursche T, Kuhlisch E, Kettner K, Grunewald S, Kratzsch J, Dittmar G, Glander HJ, Hoflack B, Kriegel TM (2011) Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis. Mol Cell Proteomics 10(8):M110 007187. https://doi.org/10.1074/mcp.M110.007187 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hooper NM (1990) Proteolytic enzymes: a practical approach Edited by R J Beynon and J S Bond. pp 259. IRL Press at Oxford University Press, Oxford. 1989. £29 (spiral bound)/£19 (paper) ISBN 0-19-963058-5/963059-3. Biochem Educ 18(1):1–60CrossRefGoogle Scholar
  8. 8.
    Noble JE, Bailey MJ (2009) Quantitation of protein. Methods Enzymol 463:73–95. https://doi.org/10.1016/s0076-6879(09)63008-1 CrossRefPubMedGoogle Scholar
  9. 9.
    Gao Y, Lui WY, Lee WM, Cheng CY (2016) Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells. Sci Rep 6:28589. https://doi.org/10.1038/srep28589 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Xiao X, Cheng CY, Mruk DD (2012) Intercellular adhesion molecule-1 is a regulator of blood-testis barrier function. J Cell Sci 125(Pt 23):5677–5689. https://doi.org/10.1242/jcs.107987 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Xiao X, Cheng CY, Mruk DD (2013) Intercellular adhesion molecule-2 is involved in apical ectoplasmic specialization dynamics during spermatogenesis in the rat. J Endocrinol 216(1):73–86. https://doi.org/10.1530/JOE-12-0434 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Goldberg S (2015) Mechanical/physical methods of cell distribution and tissue homogenization. Methods Mol Biol 1295:1–20. https://doi.org/10.1007/978-1-4939-2550-6_1 CrossRefPubMedGoogle Scholar
  13. 13.
    Hiromi Tissera MK, Stochaj U (2010) Nuclear envelopes show cell-type specific sensitivity for the permeabilization with digitonin. Protocol Exchange. https://doi.org/10.1038/protex.2010.211

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Laboratory of Signal Transduction, Medical Sciences Department, Institute for Research in BiomedicineUniversity of AveiroAveiroPortugal

Personalised recommendations