Figure 1: Structure of the Nrf2 protein.

Introduction to Nrf2

Protein Function

• Nrf2 is a crucial transcription factor that plays a key role in the response to oxidative stress. It binds to antioxidant response (ARE) element present in the promoter regions of many cell-protective genes.
• In normal conditions, Nrf2 is ubiquitinated and degraded in the cytoplasm by the BCR(KEAP1) complex.
• In response to oxidative stress, electrophile metabolites inhibit activity of the BCR(KEAP1) complex, promoting nuclear accumulation of NFE2L2/NRF2, heterodimerization with one of the small Maf proteins, leading to their accumulation in the nucleus.
• Nrf2 also be involved in the transcriptional activation of genes of the beta-globin cluster by mediating enhancer activity of hypersensitive site 2 of the beta-globin locus control region.

Protein Expression

• Broadly expressed.
• Highly expressed in adult muscle, kidney, lung, liver tissues, and neonatal muscle tissues.

Protein Localization

• Under normal conditions, Nrf2 is located in the cytoplasm.
• During oxidative stress or stimulation by electrophilic reagents and chemical activators, Nrf2 accumulates in the nucleus.

Figure 2: Nrf2 ICC Experiment Results, Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352). Green: Nrf2, Red: Tubulin, Blue: DAPI.

Isoforms & Post-Translational Modifications

• Human (Q16236): Isoforms 1-3: 65-68 kDa (predicted)
• Mouse (Q60795): 67 kDa (predicted)
• Rat (O54968): Isoforms 1-2: 67-68 kDa (predicted)
• Nrf2 undergoes acetylation, glycosylation, phosphorylation, and ubiquitination modifications.

WB Experiment Tips

Precautions

• Under normal conditions, Nrf2 is ubiquitinated and degraded in the cytoplasm, making it difficult to detect in WB. Adding MG132 can effectively inhibit this degradation process, increasing the Nrf2 detection signal.
• Oxidative stress, electrophilic reagents, chemical activators, and autophagy cause NFE2L2/NRF2 to accumulate in the nucleus. Therefore, appropriate sample treatment is crucial for the success of the WB experiment.
• The predicted molecular weight of Nrf2 is 68 kDa. However, due to isoforms and post-translational modifications, multiple bands may be observed in the WB experiment. Additionally, the actual detected molecular weight can exceed 100 kDa, differing from the predicted value. It is recommended not to cut the membrane.

Positive Control

• Whole cell lysate of Hela or HepG2 treated with 2 µM MG-132 for 18 hours
• Whole cell lysate of HCT 116 treated with 25 µM MG-132 for 4 hours
• Nuclear lysate of MDA-MB-231 treated with 30 µM tBHQ for 4 hours
• Recombinant Human Nrf2 protein (ab132356)

Negative Control

• Whole cell lysate of untreated normal HeLa, HCT 116, HepG2, etc.

Example of results:

Figure 3: WB-Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352).
Primary Antibody: ab62352, used at 1/200 dilution.
Blocking: 5% non-fat milk.

Lane 1: HCT 116 whole cell lysates
Lane 2: HCT 116 whole cell lysates treated with 25 µM MG-132 for 4 hours

Antibody: ab97051 at 1/20000 dilution.
Predicted Band Size: 68 kDa.
Detected Band Size: 100 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. Sonicate cell samples to release your target protein into solution and obtain a higher yield.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20μg total protein for electrophoresis.

Transfer:

1. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Antibody incubation:

1. It is not recommended to reuse antibodies and to always use fresh antibody preparations.

References

1. H C Huang, T Nguyen, C B Pickett. Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12475-80. doi: 10.1073/pnas.220418997.
2. Viraj R Sanghvi, Josef Leibold, Marco Mina, Prathibha Mohan, Marjan Berishaj, Zhuoning Li, Matthew M Miele, Nathalie Lailler, Chunying Zhao, Elisa de Stanchina, Agnes Viale, Leila Akkari, Scott W Lowe, Giovanni Ciriello, Ronald C Hendrickson, Hans-Guido Wendel. The Oncogenic Action of NRF2 Depends on De-glycation by Fructosamine-3-Kinase. Cell. 2019 Aug 8;178(4):807-819.e21. doi: 10.1016/j.cell.2019.07.031.
3. P Moi, K Chan, I Asunis, A Cao, Y W Kan. Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9926-30.