DIR8 Antibody

What is IRF-8 and what biological functions does it serve?

IRF-8 (Interferon Regulatory Factor 8), also known as ICSBP (Interferon Consensus Sequence Binding Protein), is a transcription factor predominantly expressed in hematopoietic cells. Its expression increases upon interferon treatment, particularly IFN-γ, but also IFN-α and IL-12 in NK and T cells . IRF-8 serves several critical biological functions:

• Functions as a transcription repressor of ICS-containing promoters

• Regulates the downregulation of anti-apoptotic protein Bcl-2

• Controls hematopoiesis (abnormal regulation resembles chronic myelogenous leukemia)

• Regulates bone metabolism by suppressing osteoclast formation

• Influences immune responses to various pathogens

IRF-8 deficient mice exhibit enhanced susceptibility to various pathogens, impaired production of interferons, and deregulated hematopoiesis that resembles chronic myelogenous leukemia .

What are the technical specifications of IRF-8 (D20D8) Rabbit mAb?

The IRF-8 (D20D8) Rabbit mAb (#5628) has the following specifications:

This antibody has been validated for multiple applications including Western Blotting (WB), Immunoprecipitation (IP), and Chromatin Immunoprecipitation (ChIP) .

What are the recommended protocols and dilutions for IRF-8 (D20D8) Rabbit mAb across different applications?

For optimal experimental results with IRF-8 (D20D8) Rabbit mAb, the following application-specific dilutions are recommended:

For ChIP and ChIP-seq experiments specifically, use 20 μl of antibody with 10 μg of chromatin (approximately 4 x 10^6 cells) per IP reaction. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits .

How should ChIP experiments be designed when using IRF-8 (D20D8) Rabbit mAb?

When designing ChIP experiments with IRF-8 (D20D8) Rabbit mAb:

• Start with the recommended dilution of 1:25

• Use 20 μl of antibody and 10 μg of chromatin (approximately 4 x 10^6 cells) per IP reaction

• Include appropriate controls:

  • Input chromatin (non-immunoprecipitated)

  • IgG control immunoprecipitation

  • Positive control regions known to bind IRF-8

  • Negative control regions with no expected IRF-8 binding

• Ensure proper crosslinking of protein-DNA complexes

• Optimize sonication conditions to generate chromatin fragments of 200-500 bp

• When analyzing results, focus on ICS-containing promoters as potential IRF-8 binding sites

This antibody has been specifically validated for ChIP applications, making it suitable for studying IRF-8's role as a transcriptional regulator in various biological contexts .

How can IRF-8 (D20D8) Rabbit mAb be utilized in studies of hematopoietic development and related disorders?

The IRF-8 (D20D8) Rabbit mAb can be instrumental in investigating hematopoiesis and related pathologies:

• Expression profiling: Track IRF-8 levels during different stages of hematopoietic differentiation using Western blotting (1:1000 dilution)

• Pathological analysis: Compare IRF-8 expression between normal samples and those from hematological disorders, particularly those resembling chronic myelogenous leukemia, which is associated with IRF-8 deficiency

• Transcriptional regulation: Use ChIP (1:25 dilution) to identify IRF-8 binding sites in hematopoietic progenitors and differentiated cells

• Protein interactions: Employ immunoprecipitation (1:50 dilution) to identify binding partners that mediate IRF-8's effects on hematopoiesis

• Mechanistic studies: Investigate the relationship between IRF-8 and Bcl-2 repression, which affects apoptotic regulation in hematopoietic cells

For comprehensive studies, combine IRF-8 detection with lineage-specific markers to correlate IRF-8 activity with specific developmental stages or pathological conditions.

What approaches can be used to study IRF-8's role in interferon signaling pathways?

To investigate IRF-8's function in interferon signaling:

• Induction studies: Monitor IRF-8 expression changes following treatment with different interferons (IFN-γ, IFN-α) and IL-12 using Western blotting (1:1000 dilution)

• Transcriptional regulation analysis:

  • Use ChIP-seq (1:25 dilution) to map genome-wide IRF-8 binding sites following interferon stimulation

  • Compare binding profiles under different stimulation conditions (IFN-γ vs. IFN-α vs. IL-12)

• Protein complex characterization:

  • Employ immunoprecipitation (1:50 dilution) to identify dynamic protein interactions following interferon stimulation

  • Investigate co-factors that determine whether IRF-8 acts as a transcriptional activator or repressor

• Functional studies:

  • Correlate IRF-8 binding with transcriptional outcomes using RNA-seq in combination with ChIP-seq

  • Investigate differential binding patterns in IRF-8 deficient models to understand pathogen susceptibility mechanisms

These approaches can help elucidate how IRF-8 contributes to the specificity and diversity of interferon responses in different cell types.

What are common challenges when using IRF-8 (D20D8) Rabbit mAb, and how can they be addressed?

Common challenges and solutions when working with IRF-8 (D20D8) Rabbit mAb include:

• Low signal intensity in Western blotting:

  • Increase antibody concentration beyond the recommended 1:1000 dilution

  • Optimize protein loading (20-30 μg total protein minimum)

  • Extend exposure time or use more sensitive detection methods

  • Verify IRF-8 expression in your sample type (remember expression is predominantly in hematopoietic cells)

  • Consider treating cells with interferons to upregulate IRF-8 expression

• Background issues in immunoprecipitation:

  • Optimize washing stringency (buffer composition, number of washes)

  • Adjust antibody amount (starting from the recommended 1:50 dilution)

  • Pre-clear lysates thoroughly

  • Use appropriate blocking agents

• Poor enrichment in ChIP experiments:

  • Verify chromatin fragmentation efficiency

  • Optimize crosslinking conditions

  • Adjust antibody:chromatin ratio (starting from 20 μl antibody to 10 μg chromatin)

  • Ensure target protein is accessible (consider epitope masking by protein-protein interactions)

• Non-specific bands in Western blotting:

  • Verify that your primary band appears at the expected 50 kDa size

  • Optimize blocking conditions

  • Consider using IRF-8 knockout or knockdown samples as negative controls

How can researchers distinguish between IRF-8 and other IRF family members in experimental data?

To ensure specificity for IRF-8 versus other IRF family members:

• Molecular weight verification:

  • IRF-8 has a molecular weight of 50 kDa , which differs from other IRF family members

  • Run appropriate molecular weight markers alongside samples

  • Consider using recombinant IRF proteins as controls

• Expression pattern analysis:

  • IRF-8 is predominantly expressed in hematopoietic cells , unlike some other IRF family members

  • Verify that signal appears in expected tissue/cell types

  • Compare expression patterns with published data on IRF family distribution

• Induction profile:

  • IRF-8 is induced by IFN-γ, IFN-α, and IL-12

  • Compare induction patterns with known profiles of other IRF family members

  • Use cytokine stimulation as a specificity control

• Functional validation:

  • In ChIP experiments, verify enrichment at known IRF-8 specific binding sites

  • Confirm association with IRF-8-specific binding partners

  • Correlate with known IRF-8-dependent functions (e.g., osteoclast formation suppression)

• Genetic validation:

  • Use IRF-8 knockout or knockdown systems as negative controls

  • Complement with IRF-8 overexpression models

What are the relative advantages of using ChIP-seq versus conventional ChIP with IRF-8 (D20D8) Rabbit mAb?

When deciding between ChIP-seq and conventional ChIP with IRF-8 (D20D8) Rabbit mAb:

Both approaches require the same antibody dilution (1:25) and amount (20 μl per reaction with 10 μg chromatin) . The choice should be guided by your research question - use ChIP-seq for discovery-based approaches and global binding pattern analysis, and conventional ChIP for hypothesis-testing on specific genes or regions.

What methodological considerations are important when comparing data from IRF-8 (D20D8) Rabbit mAb with other antibody-based techniques in interferon research?

When comparing IRF-8 (D20D8) Rabbit mAb results with other antibody-based techniques:

• Epitope accessibility:

  • The D20D8 clone recognizes a specific epitope that may be differentially accessible in various experimental conditions

  • Consider how fixation, denaturation (for WB), or crosslinking (for ChIP) might affect epitope recognition

  • Compare with antibodies targeting different IRF-8 epitopes when possible

• Antibody format considerations:

  • As a recombinant antibody, IRF-8 (D20D8) offers superior lot-to-lot consistency

  • When comparing with non-recombinant antibodies, account for potential batch variation

  • Document lot numbers for reproducibility

• Cross-reactivity assessment:

  • IRF-8 (D20D8) has confirmed reactivity with human and mouse samples

  • When comparing across species, verify species reactivity of all antibodies used

• Application optimization:

  • Each application (WB, IP, ChIP) requires specific dilutions

  • Ensure that comparison antibodies are similarly optimized for their respective applications

  • Standardize protocols when comparing antibodies in the same application

• Validation approaches:

  • Use genetic models (knockouts, knockdowns) to validate specificity

  • Compare antibody performance in interferon-stimulated versus unstimulated samples

  • Consider orthogonal detection methods (e.g., mass spectrometry) for validation

By addressing these methodological considerations, researchers can ensure robust and reproducible results when studying IRF-8's role in interferon signaling pathways.