Skip to main content

Purification of Primary Malignant B-Cells and Immunoblot Analysis of Bcl-2 Family Proteins

  • Protocol
Lymphoma

Part of the book series: Methods in Molecular Medicineā„¢ ((MIMM,volume 115))

  • 564 Accesses

Abstract

The ability to isolate relatively pure populations of primary normal and malignant lymphocytes has brought studies of lymphoid malignancies to the forefront of cancer research. Apoptosis (programmed cell death) plays a key role in controlling normal B-cell numbers, and resistance to apoptosis contributes to lymphomagenesis and reduces the effectiveness of chemotherapy and radiotherapy. Multiple Bcl-2 family proteins orchestrate key life and death decisions in lymphocytes, and the prototypical family member, Bcl-2, is activated by reciprocal translocation in human lymphoma. Here, we describe an immunomagnetic method to isolate and purify malignant B-cells suitable for in vitro analyses from lymph node biopsies. Methods to analyze the expression of Bcl-2 family proteins by immunoblotting also are described.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Cohen, J. J. (1991) Programmed cell death in the immune system. Adv. Immunol, 50, 55ā€“85.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  2. Ashkenazi, A. and Dixit, V. M. (1998) Death receptors: signaling and modulation. Science 28, 1305ā€“1308.

    ArticleĀ  Google ScholarĀ 

  3. Thornberry, N. A., and Lazebnik, Y. (1998) Caspases: enemies within. Science 28, 1312ā€“1316.

    ArticleĀ  Google ScholarĀ 

  4. Evan, G. and Littlewood, T. (1998) A matter of life and cell death. Science 28, 1317ā€“1322.

    ArticleĀ  Google ScholarĀ 

  5. Adams, J. A. and Cory, S. (1998) The Bcl-2 protein family: arbiters of cell survival. Science 28, 1322ā€“1326.

    ArticleĀ  Google ScholarĀ 

  6. Zorning, M., Heuber, A., and Baum, W. (2001) Apoptosis regulators and their role in tumorigenesis. Biochim. Biophys. Acta. 1551, F1ā€“F37.

    Google ScholarĀ 

  7. Gross, A., McDonnell, J. M., and Korsmeyer, S. J. (1999) Bcl-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 1899ā€“1911.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  8. Kroemer, G. and Reed, J. C. (2000) Mitochondrial control of cell death. Nat. Med. 6, 513ā€“519.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  9. Tsujimoto, Y. and Shimizu, S (2000) Bcl-2 family: life-or-death switch. FEBS Lett. 466, 6ā€“10.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  10. Korsmeyer, S. J. (1999) BCL-2 gene family and the regulation of programmed cell death. Cancer Res. 59, 1693ā€“1700.

    Google ScholarĀ 

  11. Adachi, M., Tefferi, A., Greipp, P.R., Kipps, T. J., and Tsujimoto, Y. (1990) Preferential linkage of bcl-2 to immunoglobulin light chain gene in chronic lymphocytic leukemia. J. Exp. Med. 171, 559ā€“564.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  12. Brimmell, M., Mendiola, R., Mangion, J., and Packham, G. (1998) BAX frameshift mutations in cell lines derived from human haemopoietic malignancies are associated with resistance to apoptosis and microsatellite instability. Oncogene 16, 1803ā€“1812.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  13. Meijerink, J. P., Mensink, E. J., Wang, K., et al. (1998) Hematopoietic malignancies demonstrate loss-of-function mutations of BAX. Blood 91, 2991ā€“2997.

    PubMedĀ  CASĀ  Google ScholarĀ 

  14. Packham, G., White, E. L., Eischen, C. M., et al. (1998) Selective regulation of Bcl-XL by a Jak kinase-dependent pathway is bypassed in murine hematopoietic malignancies. Genes Dev. 12, 2475ā€“2487.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  15. MacCarthy-Morrogh, L., Mouzakiti, A., Townsend, P., Brimmell, M., and Packham, G. (1999). Bcl-2-related proteins and cancer. Biochem. Soc. Trans. 27, 785ā€“789.

    PubMedĀ  CASĀ  Google ScholarĀ 

  16. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680ā€“685.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

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

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  18. Wolfman, J. C. and Wolfman, A. (2000). Endogenous c-N-Ras provides a steady-state anti-apoptotic signal. J. Biol. Chem. 275, 19315ā€“19323.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  19. Haldar, S., Jena, N., and Croce, C. M. (1995) Inactivation of Bcl-2 by phosphorylation. Proc. Natl. Acad. Sci. USA 92, 4507ā€“4511.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  20. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609ā€“619.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

Ā© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Dallman, C., Packham, G. (2005). Purification of Primary Malignant B-Cells and Immunoblot Analysis of Bcl-2 Family Proteins. In: Illidge, T., Johnson, P.W.M. (eds) Lymphoma. Methods in Molecular Medicineā„¢, vol 115. Humana Press. https://doi.org/10.1385/1-59259-936-2:001

Download citation

  • DOI: https://doi.org/10.1385/1-59259-936-2:001

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-159-2

  • Online ISBN: 978-1-59259-936-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics