Histone Modification Protocol

Post-translational modifications (PTMs) of histones, including methylation, acetylation, and phosphorylation, have been defined as epigenetic modifiers that can regulate chromatin structure and transcription.  Abnormal PTM patterns have been associated with various pathologies such as cancer, autoimmune disorders, and inflammatory and neurological diseases.  Methods to detect these modifications would provide useful information regarding epigenetic regulation of gene activation/silencing, histone modification-associated disease processes, and histone modification-targeted drug development.

The following protocol can be used for the ELISA-based detection of histone PTMs in samples containing native intact nucleosomes extracted from cultured mammalian cells.*  Commercially available alternatives are also presented that offer additional advantages in terms of speed, sensitivity, and specificity.

Reagent preparation

For Nuclear Isolation:

• PBS/Butyrate (PBS-B):



• Component	Concentration
  NaCl	135 mM
  KCl	2.5 mM
  Na2HPO4	8 mM
  KH2PO4	1.5 mM
  Na-butyrate	10 mM



• Lysis Buffer (LB):



• Component	Concentration
  Sucrose	250 mM
  Tris-HCl (pH 7.4)	10 mM
  Na-butyrate	10 mM
  MgCl2	4 mM
  Triton X-100	0.1%

  
  
• Wash Buffer C (WBC):


• Same composition as LB, excluding Triton X-100.


• Sucrose Cushion (SC):



• Component	Concentration
  Sucrose	30% in WBC



• Additional reagents:
• Protease inhibitors

For Nucleosome Isolation:

• Microccocal Nuclease Buffer (MNB):



• Component	Concentration
  NaPO4 buffer (pH 7.0)	5 mM
  CaCl2	0.025 mM



• Digestion Stop Solution (DSS):



• Component	Concentration
  Na-EDTA (pH 8.0)	0.5 M



• Hypotonic Solution (HS):



• Component	Concentration
  Na-EDTA	0.2 mM



• Additional reagents:
• 0.1 M CaCl2
• MNase

For Nucleosome ELISA:

• Coating Buffer (CB):



• Component	Concentration
  Na2CO3 (0.2 M)	80 ml
  NaHCO3 (0.2 M)	170 ml
  dH2O	250 ml



• Wash Buffer (WB):



• Component	Concentration
  Tween-20	0.5% in PBS



• Blocking Buffer (BB):



• Component	Concentration
  BSA	5%	in PBS
  Tween-20	0.5%



• Primary Antibody Buffer (PAB):



• Component	Concentration
  BSA	5%	in PBS
  Tween-20	0.05%



• Stop Solution (SS):



• Component	Concentration
  H2SO4	2 N



• Additional reagents:
• Primary antibody against PTM of interest
• Secondary antibody, HRP-conjugated
• TMB substrate

Histone Modification Protocol

Nuclear Isolation

1. Trypsinize cells (3 ml of trypsin per 15 cm dish).
2. Add 20 ml of ice-cold PBS-B.
3. Transfer the cell suspension to a 50 ml conical tube.
4. Centrifuge at 1000 rpm for 5 min.
5. Discard the supernatant and resuspend the cells in 10 ml of ice-cold PBS-B.
6. Centrifuge at 1000 rpm for 5 min.
7. Discard the supernatant and resuspend the cells in 4 ml of LB (+ protease inhibitors).
8. Transfer the cell suspension to a type B pestle Dounce homogenizer on ice.
9. Dounce homogenize with 20 strokes.
10. Transfer the homogenate to a centrifuge tube on ice.
11. Centrifuge at 2000×g for 10 min at 4°C.
12. Discard the supernatant and resuspend the pellet in 2 ml of ice-cold WBC (+ protease inhibitors).
13. Gently layer the resuspended material over 5 ml of SC in a cold centrifuge tube.
14. Centrifuge at 2400×g for 5 min in a swinging bucket rotor.
15. Discard the supernatant and resuspend the pelleted nuclei in 250 μl of ice-cold WBC (+ protease inhibitors).
16. Transfer the nuclei to a 1.5 mL microcentrifuge tube.

Nucleosome Isolation

1. Add 3 μl of 0.1 M CaCl2 to the nuclei.
2. Equilibrate to 37°C in a heat block.
3. Add 10 μl (2 units) of MNase (dissolved in MNB).
4. Incubate at 37°C for 12 min with frequent mixing using a pipet tip.
5. Stop MNase digestion by adding 6 μl of DSS and place on ice.
6. Centrifuge at 2000×g for 4 min, then discard the supernatant.
7. Resuspend the pellet in 300 μl of HS.
8. Incubate on ice for 1 hr with occasional gentle pipetting.
9. Centrifuge at 3000×g for 4 min at 4°C.
10. Transfer the supernatant to a fresh microcentrifuge tube on ice.
11. Repeat steps #7-#9 to free additional nucleosomes.
12. Remove the supernatant and combine with the supernatant from step 10.

Chromatin Analysis

1. Quantitation: Dilute the mononucleosome prep (e.g., 1:100) and measure absorbance at 260 nm (A260 of 10 ≈ 1 mg/ml).
2. Quality assessment: Can be analyzed via gel electrophoresis. The DNA should be predominantly 146 bp long, without laddering.
3. Storage: Aliquot and store at -80°C until ready to use.

Nucleosome ELISA

1. Prepare several dilutions of nucleosomes in CB. For example: 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.00313, 0.00156, and 0 μg (CB only), each amount per 50 μl.
2. Load each nucleosome concentration into triplicate wells (50 μl/well) of a 96-well ELISA plate and incubate with a plate cover overnight at 4°C.
3. Wash the plate 4× with 200 μl/well of WB, for 10 min total using a plate washer.
4. Block for 1 hr with 100 μl/well of BB, covered and with constant rotation.
5. Remove BB from the wells.  Cover and store the plate at -20°C for later use, or proceed immediately to step #6.
6. Add 50 μl/well of primary antibody (diluted as required in PAB).
7. Cover the plate and incubate for 1 hr, with constant rotation.
8. Repeat wash step #3.
9. Add 50 μl/well of HRP-conjugated secondary antibody (diluted as required in BB).
10. Repeat steps #7-#8.
11. Add 50 μl/well of TMB substrate. Monitor blue color development in the wells.
12. After 10 min, stop the reaction by adding 50 μl/well of SS. The blue color will turn yellow upon addition of SS.
13. Read the absorbance at 450 nm with a microplate reader.

Issues to Consider

The use of chromatin as starting material for ELISA-based histone PTM detection may be a concern, as the presence of interfering nucleic acid material can potentially hinder antibody accessibility to target modification sites.  Lengthy protocol time, low sensitivity, sample species limitations, and the need for optimization (e.g. MNase digestion conditions; choice of antibody and cross-reactivity issues; sample input amount; antibody concentration) can pose further encumbrances.

Products are available on the market that provide suitable alternatives to these issues.  Through its proprietary EpiQuik technology, EpiGentek has developed simple, rapid, and high-throughput multiplex immunoassay kits for detecting some of the most common modified histone patterns (21 and 10 different modifications for H3 and H4, respectively). These complete sets of optimized reagents are a convenient first step for quickly obtaining a broad overview of modified histones in your samples, before pursuing more in-depth analysis of particular modifications. These assays have been designed for use with histone extracts and purified histone protein isolated from a broad range of species, precluding interference from contaminating DNA. The incorporation of highly specific antibodies eliminates complications from cross-reactions and increases assay sensitivity.

*Reference Dai B, Dahmani F, Cichocki JA, Swanson LC, Rasmussen TP. Detection of post-translational modifications on native intact nucleosomes by ELISA. J Vis Exp. 2011;(50):2593. Published 2011 Apr 26. doi:10.3791/2593