Figure 1: Histone H3 protein structure.

Histone H3 Introduction

Protein Function

• Histone H3 is core component of nucleosomes.
• Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template.
• Histones play a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability.
• DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.

Protein Characteristics

• Histone H3 is core component of nucleosomes, and Sonicate cell samples to release the target protein into solution and obtain a higher yield, especially nuclear proteins.
• Histone H3 undergoes various post-translational modifications, hence the observed band size of Histone H3 may differ from the predicted band size.
• Histone H3 is a relatively small protein with a predicted molecular weight of approximately 15 kDa.

Protein Expression

• Expressed during the S phase, then expression strongly decreases as cell division slows down during the process of differentiation.

Protein Localization

• Nucleus

Figure 2: ICC Experimental Results for Histone H3, Anti-Histone H3 Antibody Product (ab176842).
Green: Histone H3; Red: Tubulin; Blue: DAPI

Isoforms & Post-Translational Modifications

• Human (P68431): 15 kDa (predicted)
  Mouse (P84228): 15 kDa (predicted)
  Rat (P84245): 15 kDa (predicted)
• Methylation (trimethylation, dimethylation, and monomethylation)
• Acetylation
• Phosphorylation
• Ubiquitination
• Glutamination

WB Experiment Tips

Precautions:

• Observed band sizes may differ from predicted sizes.
• Sonicate cell samples to release your target protein into solution and obtain a higher yield, thereby addressing no signal or weak signal issues.

Positive Controls

• Histone H3: Whole-cell lysate of HeLa (Human cervix adenocarcinoma epithelial cell) (ab150035).
• For additional positive controls, please refer to our product datasheet recommendations.

Example Results

Figure 3: WB - Anti-Histone H3 (di methyl K4) Antibody Product (ab32356)

Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2: HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3: SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
Lane 4: C6 (Rat glial tumor cell line) whole cell lysate

Predicted band size: 15 kDa
Detected band size: 17 kDa

Figure 4: WB - Anti-Histone H3 Antibody Product (ab176842)

Lane 1: Sonicated THP1 whole-cell lysate (Ultrasound time 3 s, 10 s interval, ultrasonic 5-15 times, ultrasonic power: 40 kW), 10 μg.
Lane 2: Non-sonicated THP1 whole-cell lysate, 10 μg.

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. Add a phosphatase inhibitor cocktail to prevent dephosphorylation during extraction.
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 to prohibit the degradation of the protein.
5. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. It is recommended to use a 15% separating gel for electrophoresis.
2. Load at least 20μg of total protein from cell lysate or tissue homogenate.

Transfer:

1. It is recommended to use a PVDF membrane with a pore size of 0.22μm.
2. It is recommended to use 20% methanol in the transfer buffer.
3. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Reference

1. Lowe BR, Maxham LA, Hamey JJ, Wilkins MR, & Partridge JF. Histone H3 mutations: an updated view of their role in chromatin deregulation and cancer. Cancers. (2019).11, 660. doi: 10.3390/cancers11050660.
2. Lewis, P.W., et al. Inhibition of PRC2 activity by a gain of function H3 mutation found in pediatric glioblastoma. Science (2013).340, 857–61. doi: 10.1126/science.1232245.
3. Ching Y, Wan E, Liu JX, Chan KM. Histone H3 mutations in cancer. Curr Pharmacol Rep. 2018;4(4):292-300. doi: 10.1007/s40495-018-0141-6.