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Protein Extraction from Mice Xenograft Tumor   

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Abstract

Proteins from frozen tissue such as xenograft tumor need to be extracted efficiently without being degraded to make the best use of a limited resource and to ensure that an accurate representation of proteins in selected tumor tissue can be obtained. This protocol describes how to extract proteins from xenograft tumors in mice.

Materials and Reagents

  1. NaCl
  2. Tris
  3. EDTA
  4. NP-40
  5. EGTA
  6. NaF
  7. Beta-glycerophosphate
  8. Sodium pyrophosphate
  9. Sodium vanadate
  10. Isoflurane
  11. PMSF
  12. Laemmli buffer
  13. Liquid nitrogen
  14. Phosphatase inhibitor 1/2 (Sigma-Aldrich, catalog number: P2850 , P5726 )
  15. BCA kit (Thermo Fisher Scientific, catalog number: 23227 )
  16. Lysis buffer(see Recipes)

Equipment

  1. Centrifuges
  2. Sonicator
  3. Western blotting equipment

Procedure

  1. Anesthetize the mice using isoflurane.
  2. Rapidly remove the tumor from live mice.
  3. Flash freeze the xenograft tumor in liquid nitrogen, then store at -80 °C or proceed to direct preparation of the samples.
  4. Break frozen tissue (present in liquid nitrogen) into several small pieces, and weigh one or two pieces.
  5. In dry ice, smash tissue in to smaller pieces, then add lysis buffer 2.5 μl/mg. Homogenize on ice.
  6. Sonicate for 5 sec at low setting on ice. Incubate on ice for 30 min.
  7. Spin at 13,000 rpm for 15 min at 4 °C.
  8. Transfer supernatant to a new tube.
  9. Dilute samples and perform protein assay using Pierce’s BCA kit.
  10. Adjust the concentration to an identical level.
  11. Add 5x Laemmli buffer and boil for western blot.

Recipes

  1. Lysis buffer
    150 mM NaCl
    10 mM Tris
    1 mM EDTA
    1% NP-40
    1 mM EGTA
    50 mM NaF
    40 mM beta-glycerophosphate
    10 mM sodium pyrophosphate
    1 mM sodium vanadate
    8 mM PMSF
    phosphatase inhibitor 1/2

References

  1. Dignam, J. D. (1990). Preparation of extracts from higher eukaryotes. Methods Enzymol 182: 194-203.
  2. Pang, H., Flinn, R., Patsialou, A., Wyckoff, J., Roussos, E. T., Wu, H., Pozzuto, M., Goswami, S., Condeelis, J. S., Bresnick, A. R., Segall, J. E. and Backer, J. M. (2009). Differential enhancement of breast cancer cell motility and metastasis by helical and kinase domain mutations of class IA phosphoinositide 3-kinase. Cancer Res 69(23): 8868-8876.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Pang, H. (2012). Protein Extraction from Mice Xenograft Tumor. Bio-protocol Bio101: e202. DOI: 10.21769/BioProtoc.202.
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