Skip to main content

Detection and Characterization of Apoptosis-Related Proteins in Hippocampal Neurodegeneration: From mRNA Expression to Protein Quantification

  • Protocol
  • First Online:
Neuroprotection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2761))

  • 347 Accesses

Abstract

The involvement of apoptosis in neurodegeneration can be detected by quantifying the apoptotic proteins in hippocampal lysate. Apoptosis can occur due to the overproduction of apoptotic proteins under the influence of external trigger or due to the overexpression of the apoptotic genes. Thus, the imbalance in the production of apoptotic proteins can be quantified using the Western blotting technique and the overexpression of apoptotic genes in hippocampal DNA can be quantified using the real-time quantification of mRNA expression of the apoptotic proteins. Here we provide the methodology of detecting the apoptosis-related proteins like Bax and Bcl-2 and their mRNA expression in hippocampal neurodegeneration. In this chapter, we have described the methodology for quantification of mRNA expression of these apoptosis-related proteins in the hippocampal lysate using the real-time quantitative polymerase chain reaction (qPCR) technique and the methodology of detection and characterization of respective protein expression in the hippocampal lysate using the Western blotting technique.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 219.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Okouchi M, Ekshyyan O, Maracine M, Aw TY (2007) Neuronal apoptosis in neurodegeneration. Antioxid Redox Signal 9:1059–1096

    Article  CAS  PubMed  Google Scholar 

  2. Mullis KB (1990) The unusual origin of the polymerase chain reaction. Sci Am 262:56–65

    Article  CAS  PubMed  Google Scholar 

  3. Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. In: Methods in enzymology. Elsevier, pp 335–350

    Google Scholar 

  4. Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Bio/Technology 11:1026–1030

    CAS  PubMed  Google Scholar 

  5. Valasek MA, Repa JJ (2005) The power of real-time PCR. Adv Physiol Educ 29:151–159

    Article  PubMed  Google Scholar 

  6. Hirano S (2012) Western blot analysis. Nanotox Methods Protoc:87–97

    Google Scholar 

  7. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76:4350–4354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gershoni JM, Palade GE (1983) Protein blotting: principles and applications. Anal Biochem 131:1–15

    Article  CAS  PubMed  Google Scholar 

  9. Renart J, Reiser J, Stark GR (1979) Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci 76:3116–3120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kurien BT, Scofield RH (1997) Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS–PAGE gel with multiple antigens. J Immunol Methods 205:91–94

    Article  CAS  PubMed  Google Scholar 

  11. Peferoen M, Huybrechts R, De Loof A (1982) Vacuum-blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate – polyacrylamide gels to nitrocellulose. FEBS Lett 145:369–372

    Article  CAS  Google Scholar 

  12. MacPhee DJ (2010) Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods 61:171–177

    Article  CAS  PubMed  Google Scholar 

  13. Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 25:192–205

    Article  CAS  PubMed  Google Scholar 

  14. Mergny J-L, Lacroix L (2003) Analysis of thermal melting curves. Oligonucleotides 13:515–537

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Rawat, K., Gautam, V., Sandhu, A., Saha, L. (2024). Detection and Characterization of Apoptosis-Related Proteins in Hippocampal Neurodegeneration: From mRNA Expression to Protein Quantification. In: Ray, S.K. (eds) Neuroprotection. Methods in Molecular Biology, vol 2761. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3662-6_22

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3662-6_22

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3661-9

  • Online ISBN: 978-1-0716-3662-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics