PRODUCT CODE: ET1608-28

Recombinant Phospho Nrf2(S40) Monoclonal Antibody (ET1608-28)

  • Recombinant

Applications

  • WB

  • ICC

  • IF

  • IHC-P

  • IP

  • FC

REACTIVITY

  • Human

Western blot analysis of Phospho-Nrf2(S40) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1608-28, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
Positive control: <br />
Lane 1: HepG2 cell lysate<br />
Lane 2: Raji cell lysate
  • Western blot analysis of Phospho-Nrf2(S40) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1608-28, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
Positive control: <br />
Lane 1: HepG2 cell lysate<br />
Lane 2: Raji cell lysate
  • ICC staining of Phospho-Nrf2(S40) in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1608-28, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
  • ICC staining of Phospho-Nrf2(S40) in A549 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1608-28, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
  • ICC staining of Phospho-Nrf2(S40) in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1608-28, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
  • Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-Nrf2(S40) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-28, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-Phospho-Nrf2(S40) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-28, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-Phospho-Nrf2(S40) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-28, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Flow cytometric analysis of Phospho-Nrf2(S40) was done on K562 cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1608-28, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).
Western blot analysis of Phospho-Nrf2(S40) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1608-28, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: HepG2 cell lysate
Lane 2: Raji cell lysate

Applications

  • WB

  • ICC

  • IF

  • IHC-P

  • IP

  • FC

REACTIVITY

  • Human

SPECIFICATIONS

Product Type

Recombinant Rabbit monoclonal primary

Product Name

Recombinant Phospho Nrf2(S40) Monoclonal Antibody (ET1608-28)

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding ser40 of human nrf2.

Host

Rabbit

Modification

Phospho

Modification Site

S40

Positive Control

Raji, A549, Hela, HepG2, human tonsil tissue, human kidney tissue, human breast carcinoma tissue.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

SU0334

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C or -80C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

1 ug/ul

PURIFICATION

Protein A purified.

MOLECULAR WEIGHT

90 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:1,000-1:5,000

  • ICC/IF

  • 1:50-1:200

  • IHC-P

  • 1:50-1:200

  • FC

  • 1:50-1:100

TARGET

UNIPROT #

PROTEIN NAME

Nuclear factor erythroid 2-related factor 2

GENE NAME

NFE2L2

SYNONYMS

NF-E2-related factor 2, NFE2-related factor 2, NFE2L2

SEQUENCE SIMILARITIES

Belongs to the bZIP family. CNC subfamily.

TISSUE SPECIFICITY

Widely expressed. Highest expression in adult muscle, kidney, lung, liver and in fetal muscle.

POST-TRANSLATIONAL MODIFICATION

Ubiquitinated in the cytoplasm by the BCR(KEAP1) E3 ubiquitin ligase complex leading to its degradation. In response to oxidative stress, electrophile metabolites, such as sulforaphane, modify KEAP1, leading to inhibit activity of the BCR(KEAP1) complex, promoting NFE2L2/NRF2 nuclear accumulation and activity. In response to autophagy, the BCR(KEAP1) complex is inactivated (By similarity).; Phosphorylation of Ser-40 by PKC in response to oxidative stress dissociates NFE2L2 from its cytoplasmic inhibitor KEAP1, promoting its translocation into the nucleus.; Acetylation at Lys-596 and Lys-599 increases nuclear localization whereas deacetylation by SIRT1 enhances cytoplasmic presence.; Glycation impairs transcription factor activity by preventing heterodimerization with small Maf proteins. Deglycation by FN3K restores activity.

SUBCELLULAR LOCATION

Cytoplasm, cytosol. Nucleus. Note=Cytosolic under unstressed conditions: ubiquitinated and degraded by the BCR(KEAP1) E3 ubiquitin ligase complex. Translocates into the nucleus upon induction by electrophilic agents that inactivate the BCR(KEAP1) E3 ubiquitin ligase complex.

FUNCTION

Transcription factor that plays a key role in the response to oxidative stress: binds to antioxidant response (ARE) elements present in the promoter region of many cytoprotective genes, such as phase 2 detoxifying enzymes, and promotes their expression, thereby neutralizing reactive electrophiles. In normal conditions, 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 and binding to ARE elements of cytoprotective target genes. The NFE2L2/NRF2 pathway is also activated in response to selective autophagy: autophagy promotes interaction between KEAP1 and SQSTM1/p62 and subsequent inactivation of the BCR(KEAP1) complex, leading to NFE2L2/NRF2 nuclear accumulation and expression of cytoprotective genes. May 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.

CITATIONS

  • Liu, Xingting et al.

    Lycopene ameliorates oxidative stress in the aging chicken ovary via activation of Nrf2/HO-1 pathway. | Aging [2018]