Protein Extraction from Mice Xenograft Tumor   

Abstract

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.