Briefly, potential nonspecific staining in the sections was blocked with 5% bovine serum albumin and 0.3% Triton X-100 in PBS. Mouse anti-Nrf2 (1 : 100) (Abcam, UK) or anti-NF-κB (1 : 100) (Abcam, UK) antibodies were used as primary antibodies and then followed by a secondary antibody conjugated with fluorescence (1 : 100) (Life technologies, USA). Fluorescent microscope (Leica, DMLB2, Germany) was utilized for viewing the stained sections.
Mouse anti nrf2
Manufactured by Abcam
Sourced in United Kingdom
Mouse anti-Nrf2 is a primary antibody that recognizes the Nrf2 protein. Nrf2 is a transcription factor that plays a key role in the regulation of genes involved in the antioxidant response. This antibody can be used in various laboratory applications to detect and study the Nrf2 protein.
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Lab products found in correlation
Dm lb2, by Leica (1 mentions)
Anti nf κb, by Abcam (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Mouse anti keap1, by Proteintech (1 mentions)
Whatman, by Cytiva (1 mentions)
Tcs sp2, by Leica (1 mentions)
Rabbit anti phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Bca assay, by Merck Group (1 mentions)
Amersham imager 680, by Cytiva (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Rabbit anti nrf2, by Abcam (1 mentions)
5 protocols using mouse anti nrf2
1
Immunofluorescence Staining of Nrf2 and NF-κB
2
Western Blot Analysis of Nrf2 and Keap1
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Purified protein (10 µg) was resolved on a 1.8% agarose-2% SDS gel and run at 80 V for approximately 1.5 h at room temperature in 1× TAE-0.1% SDS running buffer. The gel was transferred at room temperature to PVDF by an overnight wet transfer by gravity according to the manufacturer’s protocol (Whatman TurboBlotter Transfer System, Cytiva, Marlborough, MA, USA). The membrane was blocked with 5% skim milk in PBST (1X PBS, 1% Tween-20) and incubated with one of the following primary antibodies overnight at 4 °C at a concentration of 1:1000: mouse anti-Nrf2 (Abcam, Cambridge, UK; ab62352) or mouse anti-Keap1 (Proteintech Group, Inc, Rosemont, IL, USA;10503-2-AP). The membrane was incubated with the following Alexa Fluor 680-conjugated antibody for 1 h at room temperature at a concentration of 1:1500: goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA; A-21057). Blots were imaged using the ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
3
Immunofluorescence Analysis of pSTAT3 and Nrf-2
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pSTAT3 and Nrf-2 immunofluorescence were performed using primary antibodies such as rabbit anti-phospho-STAT3 (Tyr705) (Cell Signalling Technology, Boston, MA, USA) and mouse anti-Nrf-2 (Abcam, Cambidge, UK), respectively. The secondary antibodies for pSTAT3 and Nrf-2 were goat anti-rabbit 488 (Alexa Fluor) and donkey anti-mouse 546 (Alexa Fluor), respectively. DAPI counterstained the cell nuclei. Images of pSTAT3- and Nrf-2-immunolabelled specimens (40 ×) were taken by the confocal laser scanning microscope (TCS-SP2; Leica Microsystem GmbH, Wezlar, Germany). Immunofluorescence was performed 24, 48 and 72 h after treatment. However, given that no significant differences were observed among the three time points, only the results at 24 h are discussed.
4
Western Blot Analysis of Nrf2 in AdipoRON-Treated Mice
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Frozen contralateral and ispsilateral hemispheres from control and AdipoRON-treated mice were homogenized with a tissue lyser (Qiagen; Courtaboeuf, Cedex, France) in lysis buffer (50 mM Tris-HCl pH 9, 5 mM EDTA, 500 mM NaCl) containing a mixture of protease and phosphatase inhibitors (Pierce, Thermofischer, Waltham, MA, USA). Total protein extract concentration was evaluated using BCA assay (Sigma-Aldrich, St. Louis, MO, USA). Twenty micrograms of proteins were separated on 16% SDS polyacrylamide gel and transferred to nitrocellulose, followed by immunoblotting as described before [47 (link)]. The incubation was performed with specific antibodies: mouse anti-Nrf2 (Abcam, Les Ulis, France; REF: ab31163, 1:1000) at 4 °C overnight. The following day, membranes were washed and incubated with goat HRP-conjugated secondary antibodies (Jackson IR, Cambridge, UK; REF: 111-035-003, 1:1000) for 1 h 30 m before enhanced chemioluminescence detection, as recommended by the manufacturer with Amersham Imager 680.
5
Protein-Protein Proximity Analysis
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Duolink II Detection Kit was utilized to assess protein-protein proximity according to the manufacturer's instruction. Primary antibodies with different species were used for the detection of each protein pair.
Speci cally, Rabbit anti-NRF2 (Abcam) and mouse anti-Ubiquitin (Sapphire Bioscience) for PLA in Figure 3I and 3J; Rabbit anti-CAVIN1(rabbit, Proteintech) and mouse anti-NRF2 (Abcam) for PLA in Figure 4J and
Speci cally, Rabbit anti-NRF2 (Abcam) and mouse anti-Ubiquitin (Sapphire Bioscience) for PLA in Figure 3I and 3J; Rabbit anti-CAVIN1(rabbit, Proteintech) and mouse anti-NRF2 (Abcam) for PLA in Figure 4J and
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