Figure 1: Structure of human nucleosome core particle protein containing the H2A.X S139E variant.

gamma H2A.X Introduction

Protein Function

• Nucleosomes, which are composed of histones and DNA, wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries that require DNA as a template.
• Eukaryotic histones comprise five types: H1, H2A, H2B, H3, and H4.
• Variant histone H2A (H2AX) which replaces conventional H2A in a subset of nucleosomes.
• Ultraviolet light and other radiation can cause DNA damage. Within minutes of damage, histone H2AX is rapidly phosphorylated at the Ser139 site. Therefore, H2AX phosphorylated at Ser139, named gamma H2A.X, has been widely used as a marker for DNA double-strand breaks (DSBs) to indicate double-strand DNA damage and apoptosis.

Protein Expression

• Synthesized in G1 as well as in S-phase.

Protein Localization

• Nucleus.

Figure 2: gamma H2A.X ICC image, Alexa Fluor® 488 Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab195188).

Isoforms & Post-translational Modifications

Isoforms:

• Human (P16104): 15 kDa (predicted).
• Mouse (P27661): 15 kDa (predicted).
• Rat (D3ZXP3): 15 kDa (predicted).

Post-translational Modifications:

• Phosphorylation
• Acetylation

WB Experiment Tips

Precautions:

• Some cell lines may require induction to detect gamma H2A.X (e.g., induce Jurkat cells with staurosporine; induce HepG2 and Jurkat cells with etoposide). Please select the most appropriate induction conditions based on the sample type and antibody datasheet.
• Gamma H2A.X is a phosphorylated protein. To prevent protein degradation and dephosphorylation during extraction, we recommend adding a cocktail of protease inhibitors and phosphatase inhibitors to the lysis buffer.
• The predicted molecular weight of gamma H2A.X protein is 15 kDa. Experimental procedures should be tailored accordingly for handling small molecular-weight proteins. For details, refer to key control points.

Positive Control:

• HepG2 cell lysate – treated with etoposide

• Example Results:

Figure 3: WB - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299).

Lane 1: 20 µg HepG2 cell lysate – treated with etoposide
Lane 2: 20 µg HepG2 cell lysate – untreated

Predicted band size: 15 kDa

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. Keep samples on ice throughout the entire sample preparation process.
4. 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), it is recommended to use a 15% separating gel for electrophoresis.
2. Load at least 20 μg of total protein from cell lysate or tissue homogenate

Transfer:

1. We recommend using a PVDF membrane with a pore size of 0.22 μm for target proteins with a lower molecular weight.
2. We recommend using 20% methanol in the transfer buffer for target proteins with a lower molecular weight.
3. We recommend staining the membrane with Ponceau S after the transfer to confirm its success.

ICC Experiment Tips

Precautions

• Some cell lines may require induction to detect gamma H2A.X. Based on the sample type and antibody datasheet, choose appropriate induction conditions.
• Use positive controls and negative controls in which the primary antibody is omitted and only the secondary antibody is used to detect the non-specific binding of the secondary antibody with the sample.

Positive Controls

• Jurkat treated with etoposide (ETP).
• A549 treated with etoposide (ETP).
• HeLa treated with hydrogen peroxide (H2O2).

Example Results:

Figure 4: WB - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299).

HeLa cells (untreated and treated with H2O2).

Green: gamma H2A.X, Red: tubulin, Blue: DAPI.

Key control points

In the experiment, alongside routine issues, special attention must be given to the following key control points:

Cell Culture:

1. Cell culture preparation is essential for optimal ICC results. Before starting, it is necessary to ensure cells are healthy and growing normally.
2. Search the literature to confirm the intracellular localization of the detected protein and whether expression needs to be induced.

Sample Fixation:

1. If using aldehydes (such as 4% PFA) for fixation, it is recommended to fix at room temperature for 10-20 minutes.

Transparent:

1. If using aldehydes (such as 4% PFA) for cell fixation, it is recommended that you permeabilize with 0.1-0.25% Triton X-100 at room temperature for 10 minutes.

Blocking:

1. We recommend adding 0.3 M glycine to the blocking solution to quench autofluorescence.

Antibody Incubation:

1. We recommend incubating with the primary antibody overnight at 4°C to enhance the signal and reduce the background

References

1. Jingsong Yuan, Rachel Adamski, Junjie Chen. Focus on histone variant H2AX: to be or not to be. FEBS Lett. 2010 Sep 10;584(17)3717-24. doi:10.1016/j.febslet.2010.05.021. Epub 2010 May 21.
2. S Burma, B P Chen, M Murphy, A Kurimasa, D J Chen. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem. 2001 Nov 9;276(45):42462-7. doi: 10.1074/jbc.C100466200. Epub 2001 Sep 24.