Comparison of human Nrf2 antibodies: A tale of two proteins

Abstract

The Nrf2 transcription factor is a master regulator of the cellular defense against oxidative and electrophilic stress. An increase in Nrf2 protein levels and an accumulation of Nrf2 in the nucleus are key parts of the Nrf2 activation mechanism. The western blot technique remains the most widely used method to assess these changes. A well-characterized, specific antibody that is commercially available would greatly enhance these studies in the field. Here, an apparently highly specific Nrf2 monoclonal antibody, EP1808Y from Abcam, is compared with the most widely used Nrf2 antibodies, H-300 and C-20, both from Santa Cruz Biotechnology, in a panel of human cell lines. In addition to detecting Nrf2, EP1808Y avidly detects another protein present in two of the three cell lines tested. This protein can be mistaken for Nrf2 as it co-migrates with verified Nrf2 on two different polyacrylamide gel types. However, unlike Nrf2, its levels and cytoplasmic localization are unaffected by treatment with Nrf2 activators. The possibility that this band corresponds to a form of Nrf2 was excluded by siRNA and immunodepletion experiments. Finally, the monoclonal antibody D1Z9C from Cell Signaling was found to detect Nrf2 with the highest specificity of these four antibodies.

Korean Red Ginseng Pretreatment Protects Against Long-Term Sensorimotor Deficits After Ischemic Stroke Likely Through Nrf2

“…Our data suggested that Ginseng pretreatment was correlated with remarkable induction of Nrf2 downstream target markers, including NQO1, HO1, SOD2 and Gpx in the cortex, indicating the importance of Nrf2 activation in Ginseng’s neuroprotection. Since currently there is no effective commercial Nrf2 antibody (Lau et al, 2013 ; Kemmerer et al, 2015 ), it limits the direct measurement of Nrf2 protection. Together, our findings showed that Ginseng pretreatment protected against the progression and the extent of acute ischemic damage, attenuated acute ischemic sensorimotor deficit, and promoted long-term functional recovery, which might through Nrf2 activation.…”

Ezetimibe Prevents Ischemia/Reperfusion-Induced Oxidative Stress and Up-Regulates Nrf2/ARE and UPR Signaling Pathways

“…Western blots were performed as previously described [31] with some modifications. Cytoplasmic and nuclear extracts were prepared using the Nuclear Extraction Kit (Cayman Chemical Company, Ann Arbor, MI, USA), and the protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA).…”

Nrf2 mediates redox adaptations to exercise

“…It is worth noting that some of the earlier studies targeted a non-specific band for Nrf2 (57 kDa) in their immunoblots. Several groups have since identified the 90–110 kDa band as the biologically relevant Nrf2 target, despite the predicted molecular weight of Nrf2 being 57 kDa [38], [39].…”