Overview

  • Product name

  • Description

    Rabbit polyclonal to Nrf2
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WB, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Cow, Chimpanzee, Rhesus monkey, Gorilla, Orangutan
  • Immunogen

    Synthetic peptide corresponding to Human Nrf2 aa 100-200 conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab71889)

  • Positive control

    • This antibody gave a positive signal in the following Human Whole Cell Lysates: TE 671, Ramos, A498, Jurkat - Staurosporine Treated (24hr, 500nM), SK N SH

Properties

Applications

Our Abpromise guarantee covers the use of ab71890 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 90 kDa (predicted molecular weight: 68 kDa).Can be blocked with Human Nrf2 peptide (ab71889).
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function

    Transcription activator that binds to antioxidant response (ARE) elements in the promoter regions of target genes. Important for the coordinated up-regulation of genes in response to oxidative stress. May 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.
  • Tissue specificity

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

    Belongs to the bZIP family. CNC subfamily.
    Contains 1 bZIP domain.
  • Domain

    Acidic activation domain in the N-terminus, and DNA binding domain in the C-terminus.
  • Post-translational
    modifications

    Phosphorylation of Ser-40 by PKC in response to oxidative stress dissociates NFE2L2 from its cytoplasmic inhibitor KEAP1, promoting its translocation into the nucleus.
  • Cellular localization

    Cytoplasm > cytosol. Nucleus. Cytosolic under unstressed conditions, translocates into the nucleus upon induction by electrophilic agents.
  • Information by UniProt
  • Database links

  • Alternative names

    • erythroid derived 2 antibody
    • HEBP1 antibody
    • like 2 antibody
    • NF E2 related factor 2 antibody
    • NF-E2-related factor 2 antibody
    • NF2L2_HUMAN antibody
    • NFE2 related factor 2 antibody
    • NFE2-related factor 2 antibody
    • Nfe2l2 antibody
    • Nrf 2 antibody
    • NRF2 antibody
    • Nuclear factor (erythroid derived 2) like 2 antibody
    • Nuclear factor antibody
    • nuclear factor erythroid 2 like 2 antibody
    • Nuclear factor erythroid 2 related factor 2 antibody
    • Nuclear factor erythroid 2-related factor 2 antibody
    • Nuclear factor erythroid derived 2 like 2 antibody
    see all

Images

  • All lanes : Anti-Nrf2 antibody (ab71890) at 1 µg/ml

    Lane 1 : TE 671 (Human Rhabdomyosarcoma) Whole Cell Lysate
    Lane 2 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate
    Lane 3 : A498 (Human Kidney Carcinoma) Whole Cell Lysate
    Lane 4 : Jurkat Whole Cell Lysate - Staurosporine Treated (24hr, 500nM)
    Lane 5 : SK N SH (Human neuroblastoma) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 68 kDa
    Observed band size: 90 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 40 kDa. We are unsure as to the identity of these extra bands.



    The band observed at 90 kDa is consistent with the banding pattern observed for other commercially available antibodies to Nrf2 and is thought to correspond to the polyubiquinated form of Nrf2.
  • ICC/IF image of ab71890 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab71890, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) MCF7 cells at 5µg/ml.

References

This product has been referenced in:

  • Wu T  et al. Prevention of murine lupus nephritis by targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid. Arthritis Rheumatol 66:3129-39 (2014). Read more (PubMed: 25047252) »
See 1 Publication for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Question
Answer

The stock solution of the Optiblot DTT Reducer (10X) ab119199 in 2M. This means that once diluted 10 times this produces a 200 mM solution. Therefore if you usually use 10 mM with your samples I would suggest diluting this 200 times. The higher concentration should not really have an effect of the running of protein gels but as the pKa is around 8, in high concentration it can alter the pH somewhat which may affect the migration. We would normally suggest using 50 mM to ensure fully reducing the disulphide bonds in your samples.

Read More

Answer

Thank you once again for your enquiries. While my colleague is away, we have received some further information which I would like to forward to you.

In testing using ab62352, the HepG2 were not treated any differently when looking at Nrf2. We usually store the HepG2 lysate at -20 and use it when we need it.

The WB HepG2 sample preparation for testing with ab62352 was as follows:

Adherent cells

1 Grow cells to ˜90% confluency.
2 Wash cells twice with TBS to remove media
3 Add the appropriate volume of 1x Cell lysis buffer (ex: 3ml to a T175 flask)
4 Transfer cell lysates to Eppendorf tubes and sonicate for 10-15 seconds.
5 Spin at 14,000 rpm, 4 °C for 10 minutes.


10X Cell Lysis Buffer:
200 mM Tris-HCl (pH 7.5)
1.5 M NaCl
10 mM Na 2EDTA
10 mM EGTA
10% Triton
25 mM sodium pyrophosphate
10 mM β-glycerophosphate
10 mM Na 3VO 4 (activated)
10 μg/ml leupeptin


I hope this is helpful. Please let me know if you have any further questions.

Read More

Answer

Thank you for your reply. I can understand why you would not want to use milk blocking in this case. However, it seems that from the xxxxx datasheet of the antibody xxxx that the results you are seeing is roughly what they would expect. They obtain a band at ˜60 kDa. With our antibodies we have observed bands more towards 100 kDa. It may be worth contacting Santa Cruz to see why they expect this band size.

As I mentioned previously, I have contacted both the lab here in Cambridge and our colleagues in Epitomics in regards to how we prepare samples in order to test the anti-Nrf2 antibody [EP1808Y] ab62352 and the rabbit polyclonal ab71890.

They have both informed me that we have not used any special treatment of the samples in order to detect the signal. The HepG2 whole cell lysates produced for the antibody ab62352 was prepared following this protocol:

http://www.epitomics.com/products/support/wb

The lysis buffer used:

10X Cell Lysis Buffer:
200 mM Tris-HCl (pH 7.5)
1.5 M NaCl
10 mM Na 2EDTA
10 mM EGTA
10% Triton
25 mM sodium pyrophosphate
10 mM β-glycerophosphate
10 mM Na 3VO 4 (activated)
10 μg/ml leupeptin

It therefore seems we do not use any special treatment in isolating cell samples for the Western blot.

I hope this information has been of help. If I can be of any further assistance, please do let me know.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Lung)
Specification
Lung
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Dako Target Retrieval Solution
Permeabilization
No
Blocking step
5 minutes of peroxidase block then 10 minutes of protein block. These are ready-to-use reagents purchased from Dako as blocking agent for 15 minute(s) · Concentration: 100% · Temperature: 20°C

Mr. Antibody Solutions

Verified customer

Submitted Apr 08 2010

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