Figure 1: Structure of Heme Oxygenase 1 (HMOX1) target protein.

Introduction to Heme Oxygenase 1

Protein Function

• Catalyzes the oxidative cleavage of heme at the alpha-methene bridge carbon, released as carbon monoxide (CO), to generate biliverdin IXalpha while releasing the central heme iron chelate as ferrous iron.
• It affords protection against programmed cell death, and this cytoprotective effect relies on its ability to catabolize free heme and prevent it from sensitizing cells to undergo apoptosis.

Protein Expression

• Expressed at higher levels in renal cancer tissues compared to normal tissues (at protein level).

Protein Localization

• Endoplasmic reticulum membrane.

Figure 2: Heme Oxygenase 1 ICC image. Anti-Heme Oxygenase 1 antibody [EPR1390Y] (ab68477). Green: Heme Oxygenase 1; Blue: DAPI.

Isoforms & Post-Translational Modifications

• Human (P09601): 33 kDa (predicted)
• Mouse (P14901): 33 kDa (predicted)
• Rat (P06762): 33 kDa (predicted)

Post-Translational Modifications: phosphorylation.

WB Experiment Tips

Precautions

• Heme Oxygenase 1 exhibits expression specificity; please confirm the expression of the target protein in the sample beforehand. If unsure about the expression level, it is advisable to use a positive control.
• Stimulation or induction can increase the Heme Oxygenase 1 protein levels in certain cell lines (e.g., CoCl2 induction in HeLa cells). Refer to the product datasheet or relevant literature for specific induction conditions.
• The predicted molecular weight of Heme Oxygenase 1 is 33 kDa, but truncated forms of 28 kDa can be observed in some tissues (e.g., spleen tissue), and sometimes larger unknown bands may also be observed (see Figure 4).

Positive Control

• A549 whole cell lysate
• NIH-3T3 whole-cell lysate
• Mouse and rat spleen tissue lysate

Example Results

Figure 3: WB - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (ab68477).

Lane 1: 10 µg HEK-293 whole cell lysate
Lane 2: 10 µg HL-60 whole cell lysate
Lane 3: 10 µg HeLa whole cell lysate
Lane 4: 10 µg A549 whole cell lysate
Lane 5: 10 µg human spleen tissue lysate
Lane 6: 10 µg mouse spleen tissue lysate
Lane 7: 10 µg rat spleen tissue lysate

Predicted band size: 33 kDa
Observed band size: 32 kDa
Other description: Green, Heme Oxygenase 1; Red, GAPDH. Hek293 & HL60 presumed negative or very low expression.

Figure 4: WB - Anti-Heme Oxygenase 1 antibody [EP1391Y] (ab52947).

Lane 1: 20 µg HEK-293 whole cell lysates
Lane 2: 20 µg NIH/3T3 whole cell lysates
Lane 3: 20 µg A549 whole cell lysates
Lane 4: 20 µg mouse spleen lysates

Predicted band size: 33 kDa
Observed band size: 33 kDa
Exposure time: 120 seconds

Figure 5: WB - Anti-Heme Oxygenase 1 antibody (ab137749).

Lane 1: 30 µg untreated HeLa whole cell extracts
Lane 2:30 µg Treated HeLa whole cell extracts
Predicted band size: 33 kDa

Key Control Points

Along with regular considerations, it is essential to 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. It is recommended to use positive control.

Electrophoresis:

1. Load at least 20 μg of total protein from cell lysate or tissue homogenate.

Transfer:

1. We recommend staining the membrane with Ponceau S after the transfer to confirm its success.

References

1. A Wilks, S M Black, W L Miller, P R Ortiz de Montellano. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase. Biochemistry. 1995 Apr 4;34(13):4421-7. doi: 10.1021/bi00013a034.
2. L K Lightning, H Huang, P Moenne-Loccoz, T M Loehr, D J Schuller, T L Poulos, P R de Montellano. Disruption of an active site hydrogen bond converts human heme oxygenase-1 into a peroxidase. J Biol Chem. 2001 Apr 6;276(14):10612-9. doi: 10.1074/jbc.M010349200. Epub 2000 Dec 19.
3. Raffaella Gozzelino 1, Viktoria Jeney, Miguel P Soares. Mechanisms of cell protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol. 2010;50:323-54. doi: 10.1146/annurev.pharmtox.010909.105600.