Acid Extraction of Total Histone from Colon Cancer HCT116 Cells

Materials and Reagents

1. 6 cm plate
   
2. 1.5 ml tubes (Corning, Axygen^®, catalog number: MCT-150-C )
   
3. Human colon cancer cell line HCT116 (ATCC)
   
4. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM)
   
5. Acetone
   
6. Double-distilled H₂O (ddH₂O)
   
7. 2x SDS loading buffer (containing 200 mmol/L DTT)
   
8. Tris-HCl (pH = 8.0)
   
9. Potassium chloride (KCl)
   
10. Magnesium chloride (MgCl₂)
    
11. Dithiothreitol (DTT)
    
12. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
    
13. Sulfuric acid
    
14. Trichloroacetate (TCA) (Sigma-Aldrich, catalog number: T6399 )
    
15. Trichloroacetic acid (TCA) solution (see Recipes)
    
16. Lysis buffer(see Recipes)
    

Equipment

1. Pipettor (Eppendorf)
   
2. Centrifuge (cooled and room-temperature)(Eppendorf)
   
3. Rotator
   
4. Spectrophotometer (Bio-Rad Laboratories)
   
5. Metal bath

Procedure

An overview of the whole procedure is schematized in Figure 1.


Figure 1. Scheme of the complete procedure described in the protocol

1. Collect HCT116 cells (6 cm plate, 70-90% confluent) using any commonly used method in 1 ml ice-cold PBS and pellet (1,000 x g, 5 min, 4 °C).
   
2. Discard the supernatant, add 1 ml ice-cold PBS and pipet gently to resuspend the cells, then pellet it again (1,000 x g, 5 min, 4 °C).
   
3. Discard the supernatant, resuspend cell pellet in 400 μl lysis buffer, and incubate for 30 min with rotating at 4 °C.
   
4. Pellet the intact nuclei by spinning in cooled centrifuge (12,000 x g, 10 min, 4 °C).
   
5. Transfer the supernatant containing histones into a fresh 1.5 ml tube.
   
6. Add 133 μl TCA drop by drop to histone solution and invert the tube several times to mix the solutions (final concentration of TCA is 25%). (It is better to add one drop and invert the tube at once to mix the solutions, and then add another drop.) The solution will appear milky over time.
   
7. Incubate on ice for 30 min or overnight.
   
8. Pellet histones by spinning in cooled centrifuge (12,000 x g, 10min, 4 °C).
   
9. Carefully remove supernatant and wash histone pellet with 1 ml ice cold acetone without disturbing it. Acetone is used to remove acid from the solution without dissolving the histone pellet.
   
10. Spin down in centrifuge at 12,000 x g, 10 min at 4 °C.
    
11. Repeat steps 9 and 10 to wash histone pellet again.
    
12. Carefully remove all of the supernatant and air-dry histone pellet for 30 min at room temperature.
    
13. Dissolve histone pellet in appropriate volume of ddH₂O (for example, 100 μl is appropriate for a 6 cm plate), and transfer into fresh 1.5 ml tube.
    
14. Detect the concentration of histone solution with the spectrophotometer (using OD 280), and we may acquire 1-2 µg histone protein from a 70-90% confluent well on a 6 cm plate.
15. Add the same volume of 2x SDS loading buffer into histone solution, and boil it on the metal bath (100 °C, 5 min), then subject to Western blot.
    

Representative data

Figure 2. HCT116 cells were treated with etoposide and subjected to histone acid extraction. Immunoblotting was performed with the indicated antibodies.

Note: Relative Western blots have also been published in our paper Oncogene (Cao et al., 2016). For details, please refer to Figure 4 in Cao et al., 2016 (https://www.researchgate.net/publication/274316705_ATM-mediated_KDM2A_phosphorylation_is_required_for_the_DNA_damage_repair).

Recipes

1. Trichloroacetic acid (TCA) solution
   500 g TCA in 227 ml ddH₂O
   Store at room temperature, and protect from light
   
2. Lysis buffer
   10 mM Tris-HCl, pH 8.0
   1 mM KCl
   1.5 mM MgCl₂
   10 mM DTT (added immediately before use)
   1x protease inhibitor cocktail (added immediately before use)
   0.4 M sulfuric acid (added immediately before use)
   

Acknowledgments

This work was supported by Beijing Natural Science Foundation grant 7164305, the ‘973 Projects’ (2011CB910100, 2011CB504200 and 2013CB911000), National Natural Science Foundation of China (81222028, 81321003, 81472581, 81530074, 31570812 and 91319302), and grants (B70001) from the Ministry of Science and Technology of China. This protocol was adapted from previous work published in Cancer Research (Zhu et al., 2001), Oncogene (Cao et al., 2016) and Oncotarget (Lu et al., 2015).

References

1. Bannister, A. J. and Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Res 21(3): 381-395.
   
2. Cao, L. L., Wei, F., Du, Y., Song, B., Wang, D., Shen, C., Lu, X., Cao, Z., Yang, Q., Gao, Y., Wang, L., Zhao, Y., Wang, H., Yang, Y. and Zhu, W. G. (2016). ATM-mediated KDM2A phosphorylation is required for the DNA damage repair. Oncogene 35: 301-313.
   
3. Kouzarides T. (2007). Chromatin modifications and their function. Cell 128: 693-705.
   
4. Lu, S., Yang, Y., Du, Y., Cao, L. L., Li, M., Shen, C., Hou, T., Zhao, Y., Wang, H., Deng, D., Wang, L., He, Q. and Zhu, W. G. (2015). The transcription factor c-Fos coordinates with histone lysine-specific demethylase 2A to activate the expression of cyclooxygenase-2. Oncotarget 6(33): 34704-34717.
   
5. Strahl, B. D. and Allis, C. D. (2000). The language of covalent histone modifications. Nature 403(6765): 41-45.
   
6. Zhu, W. G., Lakshmanan, R. R., Beal, M. D. and Otterson, G. A. (2001). DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res 61(4): 1327-1333.