Figure 1: Histone H4 Target Protein Structure.

Histone H4 Introduction

Protein Function

• Histone H4 is a core component of nucleosomes, with each nucleosome composed of two identical subunits, each containing four histones: H2A, H2B, H3, and H4.
• Histones play a central role in transcription regulation, DNA repair, DNA replication, and chromosome stability.
• Histone H4K16Ac (acetylation) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. This modification not only inhibits the formation of 30-nanometer-like fibers within nucleosomes, regulating chromatin structure, but also influences interactions between non-histone proteins and chromatin fibers.
• Histone H4K20Me (methylation) leads to transcriptional repression. Chromatin remodeler protein L3MBTL1 is directly recruited when H4K20 is methylated, causing chromatin compaction. Evidence suggests that H4K20 methylation may regulate chromatin structure to ensure accurate DNA replication and participate in DNA damage response processes, maintaining genomic stability.
• Interactions between histone modifications do not act independently. For example, H3K4Me2/3 + H4K16Ac transcriptionally active homeotic genes, while H3K9Me2/3 + H4K20Me1 + H4K27Me3 + 5mC leads to X chromosome inactivation.

Protein Expression

• Widely expressed in the cell nucleus.

Protein Localization

• Cell nucleus.

Figure 2: ICC Experiment Results for Histone H4 Protein, Anti-Histone H4 Antibody (ab177840). Green: Histone H4; Red: alpha Tubulin; Blue: DAPI

Isoforms & Post-Translational Modifications

• Human (P62805): 11.3 kDa (predicted)

• Mouse (P62806): 11.3 kDa (predicted)

• Rat (P62804):

  • Isoform 1 (P62804-1): 11.3 kDa (predicted)
  • Isoform 2 (P62804-2): 2.1 kDa (predicted)

Post-Translational Modifications: Include acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, butyrylation, crotonylation, and succinylation, among others.

WB Experiment Tips

Precautions:

• Histone H4 is located in the cell nucleus, and sonication of samples helps enrich more target proteins. To ensure sufficient loading, consider extracting nuclear fractions/histones for WB experiments. Please view our western blot protocols for more information of histone extraction.
• When conducting histone WB experiments, 5% BSA is generally used as a blocking solution. Sometimes, using 3/5% skim milk can also yield satisfactory results (e.g., ab177840). Refer to the antibody product instructions for specific blocking solution recommendations.
• Detection methods for Histone H4 may vary. Use ECL, ECF, or infrared fluorescence detection. For example, apply ECL reagent to the membrane at room temperature for 3 minutes, and capture WB images at different exposure times: 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, and 5 minutes.
• Acetylation modifications of Histone H4 may require conditional induction to resolve weak or absent signals. For instance, when detecting Histone H4 (acetyl K16) using ab100463, HeLa cells may require induction treatment with 10 μg TSA.

Positive Control

• Human HeLa cell nuclear extract
• Mouse NIH/3T3 cell nuclear extract
• Recombinant Histone H4 protein

Negative control:

• Recombinant Histone H2A protein

Example Results

Figure 3: WB Results of Histone H4 Protein using Anti-Histone H4 Antibody (ab223875)

Lane 1: CTH (calf Thymus Histone) at 0.5 µg
Lane 2: HeLa Whole Cell Lysate at 20 µg
Lane 3: HeLa Nuclear Cell Lysate at 20 µg
Lane 4: NIH 3T3 Whole Cell Lysate at 20 µg
Lane 5: NIH 3T3 Nuclear Lysate at 20 µg
Lane 6: Histone H2A Recombinant Protein at 0.1 µg
Lane 7: Histone H4 Recombinant Protein at 0.1 µg

Predicted Band Size: 11 kDa
Exposure Time: 90 seconds

Figure 4: WB Results of Histone H4 (acetyl K16) Protein, Anti-Histone H4 (acetyl K16) Antibody (ab109463)

Lanes 1-2 : Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/6000 dilution
Lanes 3-4 : Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/24000 dilution
Lanes 1 & 3 : Untreated C6 (Rat glial tumor glial cell) whole cell lysate
Lanes 2 & 4 : C6 (Rat glial tumor glial cell) treated with Trichostatin A (final concentration is 500ng/ml) for 4 hours whole cell lysatePredicted band size: 11 kDa.

Exposure time: 3 minutes.

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Choose the most appropriate lysis buffer to give the highest yield of your target protein.
3. Sonicate cell samples to release your target protein into solution and obtain a higher yield.
4. Keep samples on ice throughout the entire sample preparation process.
5. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. For target proteins with smaller molecular weights (e.g., <25 kDa), use a higher gel concentration for electrophoresis.
2. For target proteins with a lower molecular weight (e.g. < 25 kDa), it is advisable not to completely run the dye front out of the gel during electrophoresis.
3. Load at least 20μg of total protein from cell lysate or tissue homogenate.
   It is recommended to use positive and negative controls.

Transfer:

1. For target proteins with a lower molecular weight, it is recommended to use 20% methanol in the transfer buffer.
2. After activating the PVDF membrane, thoroughly wash it to completely remove any residual methanol.
3. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Blocking:

1. Choose the appropriate blocking buffer according to the datasheet.

Antibody incubation:

1. Avoid the membrane drying out during the WB process.
2. Select a suitable antibody working concentration according to the product datasheet.
3. It is not recommended to reuse antibodies and to always use fresh antibody preparations.

References

1. Gregory D Bowman, Michael G PoirierGregory. Post-translational modifications of histones that influence nucleosome. Chem Rev. (2015) 115(6), 2274-2295. doi: 10.1021/cr500350x
2. Michael Shogren-Knaak, Haruhiko Ishii, Jian-Min Sun et al. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science. (2006), 311(5762), 844-847. doi: 10.1126/science.1124000
3. Stine Jørgensen, Gunnar Schotta, and Claus Storgaard Sørensen. Histone H4 Lysine 20 methylation: key player in epigenetic regulation of genomic integrity. Nucleic Acids Res.(2013) 41(5), 2797-2806. doi: 10.1093/nar/gkt012
4. Alexander J Ruthenburg, Haitao Li, Dinshaw J Patel and C David Allis. Multivalent engagement of chromatin modifications by linked binding modules. Nature Rev. Mol. Cell Biol. (2007) 8(12), 983-994. doi: 10.1038/nrm2298