Figure 1: Glutathione Peroxidase 4 protein structure.

Introduction to Glutathione Peroxidase 4

Protein Function

• It is required to prevent cells from ferroptosis, a non-apoptotic cell death resulting from an iron-dependent accumulation of lipid-reactive oxygen species.
• Sec at the active site is also essential for the survival of a specific type of parvalbumin-positive interneurons, thereby preventing fatal epileptic seizures.
• Plays a role in glutathione peroxidase in platelets in the arachidonic acid metabolism.

Protein Expression

• Predominantly present in the testes.
• Expressed in platelets (at protein level).

Protein Localization

• It is located in the mitochondrion and cytoplasm.

Figure 2: Glutathione Peroxidase 4 ICC image, Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (ab125066). Green: Glutathione Peroxidase 4; Red: Tubulin; Blue: DAPI.

Isoforms & Post-translational Modifications

• Human (P36969): Isoforms 1 and 2: 19-22 kDa (predicted)
• Mouse (O70325): Isoforms 1 and 2: 19-22 kDa, Isoform 3: 29 kDa (predicted)
• Rat (P36970): Isoforms 1 and 2: 19-22 kDa, Isoform 3: 29 kDa (predicted)

Post-translational modifications: Phosphorylation.

WB Experiment Tips

Precautions

• Glutathione Peroxidase 4 has expression specificity, and the amount of target protein varies across different samples. Some samples may show weak or no expression. If you are unsure whether your sample expresses the target protein, we strongly recommend using a positive control, such as high-expression testicular tissue.
• The actual detected protein band size is slightly lower than the predicted value. Therefore, we recommend not cutting the membrane or retaining at least 10-50 kDa for detection.
• The protein has a small molecular weight. Follow the small molecule protein experiment suggestions detailed below.

Positive Control

• HeLa cell lysate
• Human, mouse, rat testicular tissue lysate

Negative Control

• Human GPX4 knockout HeLa cell line (ab262509)/cell lysate (ab263935)

Example Results

Figure 3: Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (ab125066).

Lane 1: 20 µg HeLa whole cell lysate
Lane 2: 20 µg GPX4 knockout HeLa whole cell lysate
Lane 3: 20 µg Jurkat whole cell lysate

Predicted band size: 22 kDa
Observed band size: 20 kDa
Description: Green band is Glutathione Peroxidase 4, red band is GAPDH.

Key Control Points

In addition to the routine issues, pay special attention to the following key control points during the experiment:

Sample Preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.
4. We Strongly recommend using positive control.

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 staining the membrane with Ponceau S after the transfer to confirm its success (if using fluorescent detection, ensure thorough washing after Ponceau S staining).

Antibody Incubation:

1. Select a suitable antibody working concentration according to the product datasheet.

References

1. Wan Seok Yang, Rohitha SriRamaratnam, Matthew E Welsch, etc. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014 Jan 16;156(1-2):317-331. doi: 10.1016/j.cell.2013.12.010.
2. M Sutherland, P Shankaranarayanan, T Schewe, S Nigam. Evidence for the presence of phospholipid hydroperoxide glutathione peroxidase in human platelets: implications for its involvement in the regulatory network of the 12-lipoxygenase pathway of arachidonic acid metabolism. Biochem J. 2001 Jan 1;353(Pt 1):91-100.