IRF2 Antibody

What is IRF2 and why is it important in immunological research?

IRF2 is a member of the Interferon Regulatory Factor family of transcription factors that functions primarily as a transcriptional repressor by binding to interferon-sensitive response elements (ISREs). IRF2 competes with other IRF transcription factors such as IRF1 and IRF9 for these binding sites .

IRF2 plays multifaceted roles in immune regulation:

• Regulates type I interferon (IFN-α/β) signaling pathways

• Controls B-cell proliferation and antibody production

• Required for normal NK cell development and maturation

• Affects T cell exhaustion programs in tumor microenvironments

• Modulates inflammatory responses in macrophages

This broad involvement in immune cell function makes IRF2 a critical target for immunological research, particularly in studies of immune cell development, cancer immunology, and inflammatory disorders .

What applications are IRF2 antibodies validated for in research?

IRF2 antibodies have been validated for multiple research applications, with specific recommendations for each technique:

It is recommended to titrate antibodies in each testing system to obtain optimal results as sample-dependent variations may occur .

What species reactivity can I expect from IRF2 antibodies?

Most commercially available IRF2 antibodies demonstrate confirmed reactivity with:

• Human IRF2

• Mouse IRF2

• Rat IRF2

Some antibodies have also been cited for reactivity with chicken samples. Many antibodies are raised in rabbit hosts as polyclonal or monoclonal preparations, or in mouse hosts as monoclonal antibodies .

For recombinant rabbit monoclonal antibodies against IRF2, there is predicted reactivity with bovine samples based on sequence homology, though this may require experimental validation .

What is the molecular weight of IRF2 protein in Western blotting applications?

When using IRF2 antibodies for Western blotting, researchers should note the following specifications:

• Calculated molecular weight: 39 kDa (based on 349 amino acid sequence)

• Observed molecular weight: Approximately 50 kDa

This discrepancy between calculated and observed weights is typical for IRF2 detection and may result from post-translational modifications or structural properties of the protein .

How does IRF2 function in B-cell development and what experimental approaches best measure this?

IRF2 plays multiple roles in B-cell functions, particularly affecting B2 cells (follicular B cells) through:

• Regulation of proliferation in an IFN-α/β receptor (IFNAR)-dependent manner

• Control of antibody production via up-regulation of Blimp-1

• Influence on class switch recombination processes

Experimental approaches to measure IRF2 effects:

• Proliferation assays: IRF2-deficient B2 cells show reduced proliferation in response to anti-IgM but not LPS stimulation. This defect is IFNAR-dependent.

• Antibody production assessment: In vitro studies using LPS stimulation to measure IgM production and LPS plus IL-4 for class switch recombination studies.

• In vivo immunization models: IRF2-/- mice show impaired antibody production to T-dependent antigens, with delayed but eventual IgG production.

• Gene expression analysis: Measurement of Blimp-1 expression, which is inefficiently upregulated in IRF2-/- B cells.

These approaches have revealed that IRF2 deficiency impairs B-cell proliferation and antibody production while maintaining normal follicular helper T-cell development and germinal center formation .

What is the role of IRF2 in NK cell development and how can IRF2 antibodies be used to investigate this process?

IRF2 plays a critical cell-intrinsic role in natural killer (NK) cell development and functional maturation:

Key findings:

• IRF2 knockdown in cord blood hematopoietic stem cells (HSCs) greatly reduces NK cell numbers during differentiation

• This reduction is due to decreased proliferation rather than increased apoptosis in early developmental stages

• IRF2-deficient NK cells show impaired cytotoxicity against tumor targets and reduced cytokine secretion

• IRF2 overexpression has limited effects, suggesting endogenous expression levels are sufficient

Experimental approaches using IRF2 antibodies:

• ChIP-seq analysis: Using IRF2 antibodies to identify direct gene targets regulated by IRF2 during NK cell development

• Flow cytometry: Analyzing IRF2 expression at different stages of NK cell development

• Immunofluorescence: Investigating subcellular localization of IRF2 during NK cell activation

• Western blotting: Quantifying IRF2 expression levels in NK cell subsets

These approaches can help delineate the molecular mechanisms through which IRF2 orchestrates NK cell development and functional maturation .

How does IRF2 influence the CD8+ T cell exhaustion program in tumors and what implications does this have for cancer immunotherapy?

IRF2 serves as a critical feedback molecule that redirects interferon signals to suppress T cell responses in tumors:

Mechanistic findings:

• IRF2 is expressed by many immune cells in tumors in response to sustained interferon signaling

• CD8+ T cell-specific deletion of IRF2 prevents acquisition of T cell exhaustion programming

• IRF2-deficient CD8+ T cells maintain sustained effector functions that promote long-term tumor control

• These cells show increased responsiveness to immune checkpoint and adoptive cell therapies

• The enhanced tumor control requires continuous integration of both type I and type II interferon signals

Experimental methods to investigate IRF2 in CD8+ T cells:

• Conditional knockout models: CD8+ T cell-specific deletion of IRF2 to study cell-autonomous effects

• Tumor challenge models: Testing tumor growth control in IRF2-deficient versus wild-type mice

• Flow cytometry: Analyzing expression of activation markers (CD80, SLAMF1, Blimp1, Ki67, CD25) on IRF2-deficient CD8+ T cells

• Therapeutic response studies: Evaluating how IRF2 deficiency affects response to immune checkpoint blockade

These findings suggest that IRF2 may represent a potential target to enhance cancer control by preventing T cell exhaustion .

What is the role of IRF2 in macrophage inflammatory responses and how can it be experimentally assessed?

IRF2 functions as a negative regulator of pro-inflammatory responses in macrophages:

Key experimental findings:

• IRF2 overexpression inhibits LPS- and IFN-γ-induced expression of IL-6 and iNOS

• IRF2 silencing enhances inflammatory cytokine production

• IRF2 directly activates the promoter of Immune Response Gene 1 (IRG1)

• Through IRG1 regulation, IRF2 affects macrophage viability, migration, and apoptosis

Experimental approaches:

• Luciferase reporter assays: For determining transcriptional regulation of target genes like IRG1

• qRT-PCR and Western blotting: Measuring expression of inflammatory mediators

• Cell viability assays: IRF2 overexpression increases macrophage viability under inflammatory conditions

• Migration assays: IRF2 inhibits macrophage migration during inflammation

• Co-transfection experiments: Using IRF2 overexpression with target gene silencing to establish regulatory relationships

The table below summarizes IRF2 effects on inflammatory markers in macrophages:

These findings indicate that IRF2 has a complex regulatory role in macrophage inflammatory responses, which may have implications for inflammatory diseases .

What are the optimal conditions for immunoprecipitation using IRF2 antibodies?

For successful immunoprecipitation (IP) of IRF2, researchers should follow these methodological guidelines:

Recommended protocol:

• Antibody amount: Use 0.5-4.0 μg of IRF2 antibody per 1.0-3.0 mg of total protein lysate

• Sample preparation: Jurkat cells have been verified as a positive control for IRF2 IP

• Lysate conditions: Use of RIPA buffer or other IP-compatible lysis buffers

• Protein A/G beads: For rabbit host antibodies, protein A beads are typically more efficient

• Controls: Include appropriate IgG control (matching the host species of the antibody)

Validation data:
In HEL (Human bone marrow erythroleukemia) cell line:

• 6 μg of IRF2 antibody per mg of lysate successfully immunoprecipitated IRF2

• No signal was detected in control IgG IP lanes

• Detection was achieved with 3 seconds of chemiluminescence exposure

For optimal IP results, the storage conditions of the antibody (−20°C with 0.02% sodium azide and 50% glycerol pH 7.3) should be maintained for maximum activity .

What buffer conditions and antigen retrieval methods are recommended for IHC detection of IRF2?

For optimal immunohistochemical detection of IRF2 in tissue samples:

Recommended antigen retrieval methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Antibody dilution range: 1:20-1:200 (optimization recommended for each tissue type)

Validated tissue samples: Human colon cancer tissue has been successfully used for IRF2 detection

IHC protocol considerations:

• Fixation: Standard formalin fixation and paraffin embedding is compatible

• Blocking: BSA or serum from the species of the secondary antibody

• Detection systems: Both DAB and AEC chromogens have been used successfully

• Counterstaining: Hematoxylin provides good nuclear contrast

For antibodies stored with 0.02% sodium azide and 50% glycerol pH 7.3, it's recommended to store at −20°C, with stability for one year after shipment. Aliquoting is generally unnecessary for this storage condition .

How can ChIP-seq experiments with IRF2 antibodies be optimized for identifying IRF2 binding sites?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using IRF2 antibodies can provide valuable insights into the genome-wide binding patterns of IRF2. For optimal results:

ChIP-seq protocol recommendations:

• Cross-linking: Standard 1% formaldehyde for 10 minutes at room temperature

• Sonication conditions: Optimization to achieve chromatin fragments of 200-500 bp

• Antibody selection: Use ChIP-validated IRF2 antibodies (e.g., ab245658)

• Input control: Reserve 5-10% of pre-IP chromatin for normalization

• Cell types: Mouse bone marrow dendritic cells (BMDCs) have been successfully used

• Expected binding patterns: IRF2 binding shows peaks within specific genomic regions, such as the observed 156 kb region on chromosome 17

Data analysis considerations:

• Peak calling algorithms: MACS2 is commonly used

• Motif analysis: Look for interferon-stimulated response element (ISRE) motifs

• Integration with gene expression data: Correlate binding with expression changes

• Comparison with other IRF family members to identify unique and shared binding sites

ChIP-seq with IRF2 antibodies has successfully identified binding sites in BMDCs, showing specific peak distribution patterns that can be correlated with gene regulation events .

What are the differences between polyclonal and monoclonal IRF2 antibodies, and how should researchers choose between them?

When selecting between polyclonal and monoclonal IRF2 antibodies, researchers should consider several factors:

Polyclonal IRF2 antibodies:

• Epitope recognition: Recognize multiple epitopes on IRF2

• Sensitivity: Often provide higher sensitivity for applications like IHC and IP

• Lot-to-lot variability: May have inconsistency between production lots

• Cross-reactivity: Higher potential for cross-reactivity with related proteins

• Examples: 12525-1-AP (rabbit polyclonal)

Monoclonal IRF2 antibodies:

• Epitope recognition: Target a single epitope on IRF2

• Specificity: Typically provide higher specificity

• Consistency: Better lot-to-lot reproducibility

• Applications: Often preferred for applications requiring high specificity like ChIP-seq

• Examples: 13B2A38 (mouse IgG1, κ), B-80 H53L46 (recombinant rabbit monoclonal)

Recombinant monoclonal advantages:

• Better specificity and sensitivity

• Lot-to-lot consistency

• Animal origin-free formulations

• Broader immunoreactivity due to larger rabbit immune repertoire

Selection guidance:

• For Western blotting: Both types work well; monoclonals offer better specificity and reproducibility

• For IP: Polyclonals often perform better due to higher avidity

• For IHC/IF: Application-dependent; try both if uncertain

• For ChIP/ChIP-seq: Monoclonals generally preferred for consistent results

When choosing between mouse and rabbit host antibodies, note that rabbit antibodies often provide better sensitivity for many applications due to the larger rabbit immune repertoire .

How can researchers validate IRF2 knockdown or overexpression in experimental systems?

Proper validation of IRF2 knockdown or overexpression is crucial for experimental integrity. The following methodological approaches are recommended:

For IRF2 knockdown validation:

• Western blotting: Primary method using validated IRF2 antibodies to confirm protein reduction

  • Recommended dilution: 1:1000-1:4000

  • Expected band: ~50 kDa

  • Controls: β-actin or GAPDH as loading controls

• qRT-PCR: Confirmation of reduced IRF2 mRNA expression

  • Target: IRF2 mRNA

  • Reference genes: GAPDH, ACTB, or other stable housekeeping genes

  • Expected result: Significant reduction in IRF2 mRNA levels

• Functional assays: Validation through downstream effects

  • In macrophages: Increased IL-6 and iNOS expression upon LPS/IFN-γ stimulation

  • In NK cells: Reduced cell numbers and impaired cytotoxicity

For IRF2 overexpression validation:

• Western blotting: Confirmation of increased IRF2 protein expression

  • Recommended dilution: Same as for knockdown

  • Controls: Vector-only transfection

• qRT-PCR: Verification of increased IRF2 mRNA levels

• Functional validation:

  • In macrophages: Measurement of reduced IL-6 and iNOS, increased IRG1

  • Luciferase reporter assays: Increased activation of IRF2 target promoters

Example validation data from published studies:

• IRF2 overexpression in RAW264.7 cells resulted in significantly higher IRF2 protein and mRNA levels compared to control groups

• IRF2 knockdown via siRNA showed reduced IRF2 protein and mRNA levels

• Functional validation revealed opposite effects on inflammatory markers between knockdown and overexpression systems

What controls should be included when using IRF2 antibodies for different applications?

Proper experimental controls are essential for reliable results when using IRF2 antibodies:

Western blotting controls:

• Positive control samples: HeLa cells, COLO 320 cells, Jurkat cells, or mouse colon tissue

• Loading control: β-actin, GAPDH, or tubulin antibodies

• Molecular weight marker: To confirm the observed 50 kDa band

• Negative control: Samples known to have low/no IRF2 expression

• Validation control: IRF2 knockdown sample (when available)

Immunoprecipitation controls:

• Input sample: 5-10% of pre-IP lysate

• IgG control: Matching isotype from the same species as the IRF2 antibody

• Positive control lysate: Jurkat cells have been validated for IRF2 IP

Immunohistochemistry/Immunofluorescence controls:

• Positive control tissue: Human colon cancer tissue

• Negative control tissue: Tissue known to lack IRF2 expression

• Antibody omission control: Primary antibody replaced with buffer

• Isotype control: Matching isotype antibody at the same concentration

• Antigen competition: Pre-incubation of antibody with immunizing peptide (when available)

ChIP-seq controls:

• Input DNA: Sonicated chromatin before immunoprecipitation

• IgG control: Matching isotype from same species

• Positive control regions: Known IRF2 binding sites

• Negative control regions: Genomic regions not expected to bind IRF2

Following these control guidelines will help ensure the specificity and reliability of results obtained with IRF2 antibodies across different experimental applications .

How can researchers troubleshoot cross-reactivity issues with IRF2 antibodies?

When facing potential cross-reactivity issues with IRF2 antibodies, researchers should systematically approach troubleshooting:

Common cross-reactivity issues:
Some IRF2 antibodies (such as clone 13B2A38) have been reported to cross-react with a ~50 kDa protein of unknown origin when used at concentrations exceeding 0.1 μg/mL in Western blot applications .

Troubleshooting steps:

• Antibody dilution optimization:

  • Start with recommended dilutions (e.g., 1:1000-1:4000 for WB)

  • Perform serial dilutions to identify optimal concentration

  • Example: For clone 13B2A38, maintain concentration below 0.1 μg/mL

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Increase blocking time or concentration

  • Add 0.1-0.3% Tween-20 to reduce non-specific binding

• Specificity verification:

  • Use IRF2 knockout/knockdown samples as negative controls

  • Compare patterns with multiple antibodies targeting different IRF2 epitopes

  • Pre-absorb antibody with immunizing peptide when available

• Distinguish from other IRF family members:

  • IRF family shares structural similarities that may lead to cross-reactivity

  • Validate with IRF1, IRF9 knockout controls when possible

  • Use recombinant IRF family proteins for absorption controls

• Sample preparation considerations:

  • Optimize lysis buffers to reduce non-specific binding

  • Consider native vs. denatured conditions

  • Test different fixation methods for IHC/IF applications