IFNA6 Antibody

What is IFNA6 and why is it important in research?

Interferon alpha-6 (IFNA6) is a secreted protein of approximately 22.1 kDa molecular weight belonging to the alpha/beta interferon family . IFNA6 plays crucial roles in adaptive immune responses and B cell differentiation, making it an important target for immunological research . The protein functions through binding to cell surface receptors, activating the JAK-STAT signaling pathway, and ultimately triggering antiviral and immunomodulatory responses. As part of the Type I interferon family, IFNA6 research contributes to understanding innate immunity mechanisms relevant to viral infections, autoimmune disorders, and cancer immunotherapy.

What are the key characteristics of IFNA6 antibodies available for research?

IFNA6 antibodies are available in several formats with distinct properties:

Most IFNA6 antibodies are stored at -20°C in a solution containing PBS with glycerol (typically 50%) and preservatives like sodium azide (0.02%) . For optimal performance, avoid repeated freeze-thaw cycles, and for short-term storage and frequent use, some products can be stored at 4°C for up to one month .

How do I select the most appropriate IFNA6 antibody for my experiments?

Selection of an appropriate IFNA6 antibody should be guided by:

• Experimental application: Verify validation data for your specific application (WB, ELISA, ICC/IF, Flow Cytometry)

• Species reactivity: Ensure compatibility with your experimental model (human, mouse, rat)

• Antibody format: Consider polyclonal for broad epitope recognition or monoclonal for specific epitope targeting

• Immunogen region: Select antibodies raised against relevant regions (e.g., C-terminal vs. internal epitopes)

• Validation evidence: Review manufacturer data showing specificity in your application of interest

For complex experiments involving multiple techniques, prioritize antibodies validated across all your intended applications. The immunogen sequence information can be particularly valuable when investigating specific domains or when epitope accessibility might be affected by experimental conditions .

How do IFNA6 antibodies perform in distinguishing between IFNA6 and other interferon alpha subtypes?

Cross-reactivity is a significant concern when studying specific interferon alpha subtypes due to high sequence homology. When selecting IFNA6-specific antibodies:

• Review specificity validation data showing discrimination between IFNA6 and related subtypes

• Consider monoclonal antibodies designed against unique regions of IFNA6

• Implement proper controls including:

  • Positive controls using recombinant IFNA6

  • Negative controls using related interferon subtypes

  • Blocking experiments with peptides corresponding to the immunogen

When cross-reactivity testing data is unavailable, validate specificity independently using overexpression systems or knockdown approaches. The peptide immunization approach (amino acid range: 70-150) used in some IFNA6 antibodies targets regions with greater sequence divergence from other IFN-α subtypes .

What methodological considerations are crucial when detecting endogenous IFNA6 protein levels?

Detecting endogenous IFNA6 presents several challenges:

• Low basal expression levels in many cell types

• Transient expression following stimulation

• Potential cross-reactivity with other interferon alpha subtypes

Methodological recommendations include:

• Cell/tissue selection: Target tissues with documented IFNA6 expression (e.g., human epidermal keratinocytes as validated in WB applications)

• Stimulation protocols: Use established inducers such as viral mimics (poly(I:C)) or actual viral infection

• Temporal considerations: Optimize timing for harvest after stimulation

• Protein extraction methods: Use protocols that preserve cytokine integrity

• Sample concentration: Consider immunoprecipitation to enrich target protein prior to analysis

For Western blot applications, some researchers have successfully detected endogenous IFNA6 in HEK001 human epidermal keratinocyte cell lines and NHEK human normal epidermal keratinocytes, with bands observed at approximately 26 kDa under reducing conditions .

How can researchers validate IFNA6 antibody specificity in their experimental systems?

Comprehensive validation of IFNA6 antibody specificity should include:

• Overexpression validation: Transfect cells with IFNA6 expression constructs and confirm signal increase

• Knockdown/knockout verification: Use siRNA or CRISPR approaches to reduce IFNA6 expression and confirm signal reduction

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm consistent results

• Mass spectrometry validation: Confirm identity of immunoprecipitated proteins

Researchers should document expected molecular weight, which is approximately 22-26 kDa for IFNA6, with potential variation due to glycosylation or other post-translational modifications . For instance, when validating with transfected HEK-293 cells, Western blot should show a strong band in transfected cells compared to non-transfected controls .

What are the optimal protocols for Western blot detection of IFNA6?

For optimal Western blot detection of IFNA6:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • For secreted IFNA6, concentrate cell culture supernatants

  • Use reducing conditions for most applications

• Gel electrophoresis and transfer:

  • 12-15% SDS-PAGE gels are recommended for better resolution of the ~22-26 kDa IFNA6 protein

  • PVDF membranes often provide better results than nitrocellulose

• Antibody incubation:

  • Primary antibody dilutions: 1:500-2000 for polyclonal antibodies , 1-5 μg/mL for monoclonals

  • Blocking buffer: 5% non-fat dry milk in TBST has been validated

  • Secondary antibody: HRP-conjugated anti-species antibody at 1:1000-1:5000

• Detection:

  • Enhanced chemiluminescence systems provide sensitivity for detecting low-abundance IFNA6

  • Expected band size: 22-26 kDa (the slight variation may result from post-translational modifications)

Positive control lysates shown to express IFNA6 include U937 human histiocytic lymphoma cells, human fetal liver tissue, human fetal lung tissue , and human epidermal keratinocytes .

How should researchers optimize ELISA protocols for IFNA6 detection?

Optimizing ELISA protocols for IFNA6 detection requires:

• Antibody selection:

  • Capture antibody: Monoclonal antibodies often provide higher specificity

  • Detection antibody: Polyclonal antibodies can amplify signal through recognition of multiple epitopes

• Dilution optimization:

  • Typical polyclonal antibody dilutions: 1:5000-20000

  • Determine optimal concentration through titration experiments

• Sample preparation:

  • Cell culture supernatants: Test both concentrated and unconcentrated

  • Serum/plasma: Dilute appropriately to minimize matrix effects

  • Consider adding protease inhibitors to preserve cytokine integrity

• Standard curve preparation:

  • Use recombinant human IFNA6 for accurate quantification

  • Prepare fresh standards or use single-use aliquots

• Validation controls:

  • Include positive controls (samples with known IFNA6 expression)

  • Include negative controls (samples without IFNA6)

  • Spike known amounts of recombinant IFNA6 into sample matrix to assess recovery

For indirect ELISA, monoclonal antibodies like clone 3C9 have been demonstrated to be suitable . For sandwich ELISA, combinations of monoclonal capture and polyclonal detection antibodies often provide optimal sensitivity and specificity.

What are the key considerations for immunocytochemistry and flow cytometry applications with IFNA6 antibodies?

For successful immunocytochemistry (ICC) and flow cytometry applications:

• Fixation and permeabilization:

  • For intracellular IFNA6: 4% paraformaldehyde fixation followed by 0.1-0.5% Triton X-100 permeabilization

  • For secretory pathway visualization: Consider gentler permeabilization with saponin

• Antibody selection:

  • Not all IFNA6 antibodies work equally well for ICC/IF

  • Recombinant monoclonal antibodies have demonstrated suitability for ICC/IF and flow cytometry (intracellular)

• Signal amplification:

  • Consider tyramide signal amplification for low-abundance detection

  • Longer primary antibody incubation (overnight at 4°C) may improve signal

• Flow cytometry-specific considerations:

  • Stimulate cells appropriately to induce IFNA6 expression

  • Include protein transport inhibitors (e.g., Brefeldin A) during stimulation to retain intracellular cytokines

  • Test multiple permeabilization protocols if initial results are suboptimal

• Controls:

  • Include isotype controls at matching concentrations

  • Include positive control cells with known IFNA6 expression

  • Consider blocking peptide controls to confirm specificity

For flow cytometry, recombinant antibodies that have been specifically validated for intracellular applications should be prioritized .

What are common challenges in IFNA6 detection and how can they be addressed?

When troubleshooting IFNA6 detection:

For antibodies validated in transfected systems but showing poor results with endogenous protein, consider concentrating samples or using signal enhancement methods. When necessary, validate specificity through knockout/knockdown approaches or peptide competition assays.

How do differences in antibody production methods impact experimental outcomes?

Different antibody production methods yield reagents with distinct characteristics:

• Polyclonal antibodies:

  • Advantages: Recognize multiple epitopes, robust to minor protein modifications

  • Limitations: Batch-to-batch variability, potential cross-reactivity

  • Best applications: Western blot, ELISA

• Monoclonal antibodies:

  • Advantages: Consistent performance, high specificity for a single epitope

  • Limitations: May be sensitive to epitope modifications or masking

  • Best applications: Flow cytometry, high-specificity assays

• Recombinant monoclonal antibodies:

  • Advantages: Reproducibility across batches, defined sequence

  • Applications: Particularly valuable for ICC/IF, flow cytometry

• Immunogen selection impact:

  • Peptide immunogens (e.g., amino acids 70-150) may yield antibodies with different specificity compared to full-length protein immunogens

  • C-terminal targeted antibodies may perform differently than those targeting internal regions

How should researchers interpret conflicting results when using different IFNA6 antibodies?

When faced with discrepant results from different IFNA6 antibodies:

• Examine epitope differences:

  • Antibodies targeting different regions may yield different results due to:

    • Protein conformation in the experimental system

    • Post-translational modifications affecting epitope accessibility

    • Protein-protein interactions masking epitopes

• Consider methodological variables:

  • Sample preparation differences (reducing vs. non-reducing conditions)

  • Detection methods (chemiluminescence vs. fluorescence)

  • Blocking reagents and buffers

• Validate with orthogonal approaches:

  • mRNA expression analysis (qPCR)

  • Mass spectrometry identification

  • Functional assays for IFNA6 activity

• Implement systematic controls:

  • Side-by-side comparison using identical samples

  • Include recombinant IFNA6 standards

  • Test antibodies on IFNA6-overexpressing and knockdown systems

How can IFNA6 antibodies be used in studying interferon dynamics during viral infections?

IFNA6 antibodies enable researchers to:

• Track temporal expression patterns:

  • Monitor IFNA6 production kinetics after viral challenge

  • Compare IFNA6 induction patterns across different viral infections

  • Correlate IFNA6 expression with viral clearance or persistence

• Identify cellular sources:

  • Combine with cell-specific markers for flow cytometry or immunohistochemistry

  • Quantify cell type-specific contributions to IFNA6 production

  • Track changes in producing cell populations over infection course

• Investigate regulatory mechanisms:

  • Study post-transcriptional regulation by comparing mRNA and protein levels

  • Examine subcellular localization and trafficking

  • Assess IFNA6 secretion dynamics in response to different stimuli

What approaches can be used to study IFNA6-specific functions distinct from other interferon alpha subtypes?

To dissect IFNA6-specific functions:

• Neutralization experiments:

  • Use IFNA6-specific neutralizing antibodies to selectively block function

  • Compare with pan-IFN-α neutralizing approaches

  • Assess impact on downstream signaling and biological effects

• Complementary molecular approaches:

  • IFNA6-specific siRNA knockdown

  • CRISPR/Cas9 gene editing to create IFNA6-deficient cell lines

  • Selective expression of IFNA6 in interferon-deficient systems

• Receptor binding and signaling studies:

  • Investigate IFNA6-specific receptor binding characteristics

  • Examine downstream signaling pathway activation patterns

  • Compare gene expression profiles induced by IFNA6 versus other subtypes

• Structure-function analyses:

  • Use antibodies recognizing different IFNA6 domains to block functional regions

  • Correlate structural features with functional outcomes

These approaches can help determine whether IFNA6 possesses unique immunomodulatory or antiviral properties distinct from other interferon alpha subtypes, which remains an important question in interferon biology research.