NFE2L3 Antibody, HRP conjugated

What is NFE2L3 and why are NFE2L3 antibodies important for research?

NFE2L3 (also known as Nrf3) is a 694-amino acid protein encoded by the NFE2L3 gene in humans. It functions as a bZIP transcription factor involved in transcription by RNA polymerase II and is primarily localized to the nucleus . NFE2L3 plays significant roles in multiple cellular processes including carcinogenesis, stress response, differentiation, and inflammatory processes .

Antibodies against NFE2L3 are crucial research tools because they enable specific detection of this protein in complex biological samples. They allow researchers to study NFE2L3's expression patterns, subcellular localization, protein-protein interactions, and post-translational modifications. Without these antibodies, investigating the biological functions and regulatory mechanisms of NFE2L3 would be extremely challenging.

What is an HRP-conjugated NFE2L3 antibody?

An HRP-conjugated NFE2L3 antibody is a specialized immunological reagent where the antibody specific to NFE2L3 is chemically linked to horseradish peroxidase (HRP) enzyme. This conjugation creates a direct detection system that eliminates the need for secondary antibodies in many applications . When using HRP-conjugated antibodies:

• The antibody portion binds specifically to the NFE2L3 protein

• The conjugated HRP enzyme catalyzes a colorimetric, chemiluminescent, or fluorescent reaction when exposed to appropriate substrates

• This reaction produces a detectable signal proportional to the amount of NFE2L3 present

This design significantly reduces experimental time, minimizes background signal, and allows for more streamlined protocols in techniques like Western blotting, ELISA, and immunohistochemistry.

What are the common applications for NFE2L3 antibody, HRP conjugated?

NFE2L3 antibodies, including HRP-conjugated variants, are utilized in several key immunodetection techniques:

Research indicates that these applications have been instrumental in characterizing NFE2L3's involvement in cellular processes including oxidative stress response and cancer progression .

What tissues and cell types express NFE2L3?

NFE2L3 exhibits a specific expression pattern across different tissues and cell types:

• Highly expressed in human placenta

• Notable expression in B-cell and monocyte cell lines

• Present in various cancer cell lines, particularly HCT116 colon cancer cells

• Expressed in HEK293T cells (commonly used for transfection studies)

Understanding this expression profile is crucial when selecting appropriate experimental models for NFE2L3 research. When using NFE2L3 antibodies for detection in tissues with low expression, protocol optimization may be necessary to enhance sensitivity.

How is NFE2L3 regulated in cells?

NFE2L3 is tightly regulated through several mechanisms:

• Post-translational regulation through the ubiquitin-proteasome pathway

• Very short half-life (approximately 20-40 minutes), indicating rapid turnover

• Stabilized by proteasomal inhibitors such as MG-132, epoxomicin, or β-lactacystin

• Polyubiquitinated via both Lys-48 and Lys-63 ubiquitin linkages

• Regulated by F-box/WD repeat-containing protein 7 (FBW7), an E3 ubiquitin ligase

• Requires phosphorylation by glycogen synthase kinase 3 (GSK3) prior to FBW7-mediated degradation

• Contains three potential ubiquitination sites: Lys-77, Lys-219, and Lys-589, with Lys-77 appearing most significant

This complex regulation makes NFE2L3 a fascinating but challenging protein to study, highlighting the importance of specific and sensitive detection methods like HRP-conjugated antibodies.

What are the best experimental conditions for using NFE2L3 antibody, HRP conjugated in Western blotting?

For optimal results with NFE2L3 antibody (HRP conjugated) in Western blotting, consider these methodological recommendations:

• Sample preparation:

  • Include proteasome inhibitors (e.g., MG-132 at 10μM for 4-6 hours) in your cell culture prior to lysis to stabilize NFE2L3, which has a very short half-life (20-40 minutes)

  • Use a lysis buffer containing 10mM Tris-HCl (pH 8.0), 150mM NaCl, 250mM sucrose, 2mM MgCl₂, and 1% Triton X-100

  • Add phosphatase inhibitors if studying phosphorylation-dependent regulation by GSK3

• Gel electrophoresis considerations:

  • Use 8-10% polyacrylamide gels to properly resolve the 694-amino acid NFE2L3 protein

  • Expected molecular weight varies due to post-translational modifications (multiple forms labeled A, B, and C have been observed)

• Transfer and detection:

  • Transfer to PVDF membrane at 100V for 60-90 minutes with precooled transfer buffer

  • Blocking with 5% non-fat milk for 1 hour at room temperature

  • Typical dilution range: 1:1000 to 1:5000 (verify optimal concentration for your specific antibody)

  • Incubate with membrane for 2 hours at room temperature or overnight at 4°C

• Controls:

  • Include lysates from cells with known NFE2L3 expression (e.g., HCT116 cells, placental tissue extracts)

  • Consider including samples treated with and without proteasome inhibitors as positive controls

These conditions will help ensure specific detection of NFE2L3 while accounting for its rapid turnover characteristics.

How can I optimize immunohistochemistry protocols using NFE2L3 antibody, HRP conjugated?

Optimizing IHC with HRP-conjugated NFE2L3 antibody requires careful consideration of multiple parameters:

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Compare both methods to determine which best exposes the NFE2L3 epitope recognized by your antibody

  • Typically heat at 95-100°C for 15-20 minutes followed by 20-minute cooling

• Blocking endogenous peroxidase and preventing non-specific binding:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Include protein blocker (e.g., 1% BSA) to reduce background

• Antibody dilution and incubation:

  • Test a dilution series (e.g., 1:100, 1:200, 1:500) to determine optimal concentration

  • Incubate at 4°C overnight for maximum sensitivity or at room temperature for 1-2 hours

  • Consider using humidity chambers to prevent tissue drying

• Signal development and counterstaining:

  • Use DAB substrate for standard brown visualization of HRP activity

  • Monitor reaction development microscopically to prevent overdevelopment

  • Counterstain with hematoxylin to provide tissue context

  • Consider controls with tissues known to express NFE2L3, such as placenta

• Special considerations for NFE2L3:

  • As NFE2L3 is primarily nuclear, evaluate nuclear staining patterns carefully

  • Note that NFE2L3 has a short half-life, so tissue fixation timing may impact detection levels

What controls should I use when working with NFE2L3 antibody, HRP conjugated?

Appropriate controls are essential for validating results with NFE2L3 antibodies:

• Positive controls:

  • Tissues with confirmed NFE2L3 expression (placenta is particularly recommended)

  • Cell lines with known high NFE2L3 expression (HCT116, B-cell lines, monocyte lines)

  • Cells transfected with NFE2L3 expression vector (often used in mechanistic studies)

• Negative controls:

  • Isotype controls (non-specific antibody of same isotype and concentration)

  • Secondary antibody-only controls (omit primary NFE2L3 antibody)

  • Tissues or cells with confirmed absence or very low expression of NFE2L3

• Validation controls:

  • siRNA or shRNA knockdown of NFE2L3 to confirm antibody specificity

  • Preabsorption with immunizing peptide if available

  • Comparison with a non-HRP conjugated NFE2L3 antibody to confirm consistent staining patterns

• Treatment controls:

  • Cells treated with and without proteasome inhibitors (e.g., MG-132) to show stabilization of NFE2L3

  • Samples with and without phosphatase treatment if studying phosphorylation-dependent regulation

Systematic use of these controls ensures confidence in your experimental findings and helps troubleshoot any unexpected results.

How do I troubleshoot non-specific binding with NFE2L3 antibody, HRP conjugated?

Non-specific binding is a common challenge when working with antibodies. Here are systematic troubleshooting approaches:

• Common sources of non-specific binding and their solutions:

• NFE2L3-specific considerations:

  • Be aware that multiple forms of NFE2L3 (labeled A, B, and C) have been observed in some cell types

  • NFE2L3's short half-life may lead to variability between samples; standardize sample collection conditions

  • Proteasome inhibition can dramatically affect NFE2L3 levels, potentially changing banding patterns

• HRP conjugate-specific troubleshooting:

  • High background may result from endogenous peroxidase activity; ensure thorough quenching

  • Store HRP-conjugated antibodies according to manufacturer recommendations to maintain enzyme activity

  • Avoid sodium azide in buffers used with HRP-conjugated antibodies as it inhibits HRP activity

Can NFE2L3 antibody, HRP conjugated be used to study the relationship between NFE2L3 and the ubiquitin-proteasome pathway?

Yes, HRP-conjugated NFE2L3 antibodies are valuable tools for investigating NFE2L3's regulation by the ubiquitin-proteasome pathway. Here's a methodological approach:

• Experimental design for studying NFE2L3 ubiquitination:

  • Transfect cells with tagged ubiquitin constructs (e.g., HA-ubiquitin) and NFE2L3

  • Treat cells with proteasome inhibitors (e.g., MG-132) to stabilize ubiquitinated proteins

  • Perform immunoprecipitation using NFE2L3 antibody

  • Analyze immunoprecipitates by Western blot using anti-HA antibody to detect ubiquitinated NFE2L3

• Investigating FBW7-mediated regulation:

  • Co-transfect cells with NFE2L3 and FBW7 expression vectors

  • Alternatively, use FBW7 knockdown approaches using specific shRNAs

  • Perform cycloheximide chase assays to monitor NFE2L3 protein stability

  • Western blot with HRP-conjugated NFE2L3 antibody to track protein levels over time

• Studying GSK3-dependent phosphorylation:

  • Treat cells with GSK3 inhibitors (e.g., CHIR99021)

  • Use phosphatase treatment of lysates as controls

  • Detect changes in NFE2L3 stability and ubiquitination status

  • Consider using phospho-specific antibodies if available

Research has established that NFE2L3 has a very short half-life (20-40 minutes) and is stabilized by proteasomal inhibitors . The protein undergoes both Lys-48 and Lys-63 linked polyubiquitination, with a significant ubiquitination site at Lys-77 .

How can I design experiments to study NFE2L3 regulation by FBW7 and GSK3?

Based on published research, here's a comprehensive experimental approach to study this regulatory mechanism:

• Co-immunoprecipitation studies:

  • Transfect HEK293T cells with HA-tagged FBW7 and NFE2L3 expression vectors

  • Treat cells with MG-132 to prevent degradation of the interaction complex

  • Perform immunoprecipitation with NFE2L3 antibodies

  • Western blot with anti-HA antibody to detect FBW7 association

  • Include preimmune serum as negative control

• Ubiquitination assays:

  • Co-transfect cells with NFE2L3, HA-tagged ubiquitin, and either FBW7 or empty vector

  • Immunoprecipitate NFE2L3 and blot with anti-HA to detect ubiquitination

  • Compare ubiquitination levels with and without FBW7 overexpression

  • Alternatively, use FBW7 knockdown to demonstrate reduced ubiquitination

• Protein stability assays:

  • Perform cycloheximide chase experiments to track NFE2L3 degradation rate

  • Compare stability in the presence or absence of FBW7 (overexpression or knockdown)

  • Include GSK3 inhibitors to demonstrate the requirement for GSK3-mediated phosphorylation

  • Use phosphorylation-site mutants of NFE2L3 to confirm the specific sites involved

• Functional consequences:

  • Examine the effect of FBW7 on NFE2L3-mediated gene repression, particularly focusing on the antioxidant response element (ARE) of NAD(P)H:quinone oxidoreductase 1 (NQO1) gene

  • Use reporter assays with ARE-containing promoter constructs

  • Measure expression of endogenous NFE2L3 target genes

These approaches collectively provide a comprehensive framework for investigating the regulatory mechanisms controlling NFE2L3 stability and function.

What are the considerations for using NFE2L3 antibody, HRP conjugated in cancer research?

NFE2L3 has been implicated in carcinogenesis, making it a relevant target for cancer research. Here are methodological considerations when using HRP-conjugated NFE2L3 antibodies in cancer studies:

• Selection of appropriate cancer models:

  • HCT116 colon cancer cells are documented to express high levels of NFE2L3

  • Consider cells derived from placenta (e.g., choriocarcinoma) given NFE2L3's high expression there

  • Include both cancer cell lines and matched normal cells to determine cancer-specific alterations

• Experimental approaches for cancer-related studies:

  • Analyze NFE2L3 expression across cancer progression stages using tissue microarrays

  • Examine correlation between NFE2L3 levels and patient outcomes using immunohistochemistry

  • Study the impact of NFE2L3 on cancer cell proliferation, migration, and invasion through gain/loss-of-function approaches

  • Investigate interactions between NFE2L3 and known cancer pathways, particularly those involving oxidative stress

• Technical considerations:

  • When working with tumor tissue, consider heterogeneity within samples

  • Use laser capture microdissection to isolate specific cell populations if necessary

  • Be aware that fixation methods and times can affect epitope availability in clinical samples

  • For prognostic studies, develop and validate a standardized scoring system for NFE2L3 staining

• Mechanistic investigations:

  • Explore the connection between NFE2L3 and FBW7 (a known tumor suppressor) in cancer contexts

  • Examine potential alterations in NFE2L3 stability in cancer cells

  • Investigate NFE2L3's role in the antioxidant response pathway in the context of cancer metabolism

  • Consider the relationship between NFE2L3 and inflammatory processes, which are often dysregulated in cancer

NFE2L3's involvement in cellular stress responses and its regulation by the tumor suppressor FBW7 suggest it may play important roles in cancer biology that warrant further investigation .

What emerging applications exist for NFE2L3 antibody, HRP conjugated?

Several innovative applications for HRP-conjugated NFE2L3 antibodies are emerging in the research landscape:

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing):

  • Mapping genome-wide binding sites of NFE2L3 to identify novel target genes

  • Requires careful optimization of crosslinking and sonication conditions

  • Consider using cells treated with proteasome inhibitors to stabilize NFE2L3 binding

• Multiplex immunofluorescence:

  • Using tyramide signal amplification with HRP-conjugated antibodies for multiplexed detection

  • Allows co-localization studies of NFE2L3 with other proteins in the ubiquitin-proteasome pathway

  • Sequential detection of multiple targets on the same tissue section

• Single-cell applications:

  • Adapting NFE2L3 detection for microfluidic or droplet-based single-cell protein analysis

  • Investigating cell-to-cell variability in NFE2L3 expression and regulation

  • Correlation with single-cell transcriptomics data

• Therapeutic development:

  • Using NFE2L3 antibodies to validate it as a potential drug target

  • Screening compounds that modulate NFE2L3 stability or function

  • Developing antibody-drug conjugates targeting NFE2L3-expressing cells

The continued development of more specific and sensitive NFE2L3 antibodies will further enhance these applications and open new avenues for research into this important transcription factor.

How can I validate a new lot of NFE2L3 antibody, HRP conjugated?

Thorough validation ensures consistent, reliable results when using a new antibody lot:

• Comprehensive validation protocol:

• Application-specific validation:

  • For IHC/ICC: Compare staining pattern and intensity between lots

  • For IP: Confirm ability to immunoprecipitate NFE2L3 protein

  • For ELISA: Generate standard curves with both lots and compare sensitivity and dynamic range

• Functional validation:

  • Confirm detection of changes in NFE2L3 levels after treatment with proteasome inhibitors

  • Verify detection of changes after FBW7 overexpression or knockdown

  • Document lot-specific optimal conditions for future reference

Rigorous validation ensures experimental continuity and reliable results when transitioning to a new antibody lot.