ABCF1 Antibody

What is ABCF1 and why is it important in research?

ABCF1 is a unique ATP Binding Cassette (ABC) transporter family member that lacks a transmembrane domain and functions as a cytosolic nucleic acid sensor. It regulates CXCL10, interferon-β expression, and downstream type I interferon responses in human airway epithelial cells. ABCF1 is particularly significant in immunology research because it modulates TLR signaling and plays a multifactorial role in innate immunity . Additionally, ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain interactions .

What are the typical applications for ABCF1 antibodies in research?

ABCF1 antibodies can be utilized in multiple experimental applications:

These applications have been validated in various cell lines including HeLa, K-562, and HepG2 cells for WB, with positive IP detection in K-562 cells and positive IHC in human stomach cancer tissue .

What is the expected molecular weight for ABCF1 in Western blot applications?

The predicted molecular weight for ABCF1 is approximately 95 kDa, but the observed band size in Western blots is typically around 85 kDa . This discrepancy between predicted and observed sizes is important to note during experimental validation. Researchers should be aware that protein migration patterns can be affected by post-translational modifications, protein folding, or the presence of different isoforms.

How does ABCF1 function in the innate immune response to viral infections?

ABCF1 functions as a cytosolic nucleic acid sensor in human airway epithelial cells, playing a critical role in the detection of double-stranded DNA (dsDNA) viral mimics. Upon recognition of dsDNA, ABCF1 mediates the production of CXCL10, a chemokine involved in recruiting immune cells to sites of infection. Knockdown experiments have demonstrated that reduced ABCF1 expression attenuates select genes involved in antiviral responses .

Gene Ontology analyses reveal that ABCF1 has a broader interaction with TLR signaling pathways beyond just nucleic acid sensing. This suggests a multifaceted role in coordinating innate immune responses in human airway epithelial cells. The precise mechanism of action likely involves IRF-3 (Interferon Regulatory Factor 3) and subsequent production of CXCL10, though the exact molecular interactions remain an active area of research .

What are the known isoforms of ABCF1 and how do they differ functionally?

Multiple isoforms of ABCF1 have been identified, with specific functional differences:

Isoform 2 is particularly well-characterized and is required for efficient Cap- and IRES-mediated mRNA translation initiation. Notably, this isoform is not involved in ribosome biogenesis . This functional specificity makes isoform 2 particularly interesting for researchers studying translation regulation.

The differential roles of ABCF1 isoforms highlight the importance of using antibodies that can distinguish between these variants or designing experiments that account for isoform-specific functions when studying ABCF1 in different cellular contexts.

How does ABCF1 expression vary across different tissue types and in disease states?

ABCF1 gene and protein expression has been confirmed in human lung tissue and human airway epithelial cells . When designing studies to investigate ABCF1 expression across different tissues or in disease states, researchers should consider:

• Baseline expression levels in normal tissues

• Changes in expression during inflammatory responses

• Potential alterations in expression in disease states such as respiratory infections or chronic inflammatory conditions

Immunohistochemistry studies have shown positive ABCF1 detection in human stomach cancer tissue , suggesting potential roles in cancer biology that warrant further investigation.

What are the optimal conditions for using ABCF1 antibodies in immunohistochemistry?

For successful immunohistochemistry with ABCF1 antibodies, the following protocol considerations are crucial:

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal results. Alternatively, citrate buffer at pH 6.0 may be used, though this might yield different staining intensities .

• Fixation: Standard formalin fixation protocols are generally compatible with ABCF1 antibodies, but excessive fixation should be avoided as it may mask epitopes.

• Controls: Always include positive controls (such as human stomach cancer tissue) and negative controls to validate staining specificity.

• Optimization: Antibody concentration should be optimized for each specific tissue type, as background staining can vary depending on tissue composition and processing methods.

How can researchers validate ABCF1 antibody specificity for experimental applications?

Validation of ABCF1 antibody specificity is critical for experimental reliability. A comprehensive validation approach should include:

• Western blot analysis: Confirm single band detection at the expected molecular weight (approximately 85 kDa observed vs. 95 kDa predicted) .

• Knockdown/knockout controls: Use siRNA-mediated knockdown or CRISPR-based knockout of ABCF1 to confirm antibody specificity by demonstrating reduced or absent signal.

• Multiple antibody validation: When possible, use antibodies from different vendors or those targeting different epitopes of ABCF1 to confirm staining patterns.

• Recombinant protein controls: Use purified recombinant ABCF1 protein as a positive control, and unrelated recombinant proteins as negative controls.

• Cell type controls: Test antibody performance across multiple cell lines with known ABCF1 expression profiles, such as HeLa, K-562, and HepG2 cells .

What flow cytometry panel design considerations are important when including ABCF1 antibody?

When designing a flow cytometry panel that includes ABCF1 antibody, consider the following factors:

How can researchers troubleshoot inconsistent results when using ABCF1 antibodies in functional studies?

When encountering inconsistent results with ABCF1 antibodies in functional studies like siRNA knockdown experiments, consider these troubleshooting approaches:

• Validate knockdown efficiency: Confirm ABCF1 protein reduction using Western blot and quantify the degree of knockdown. Partial knockdown may lead to variable functional outcomes.

• Assess cell-type specific effects: ABCF1 functions may vary depending on cell type. Results obtained in one cell type (e.g., human airway epithelial cells) may differ from those in another context.

• Consider redundancy: Other ABC transporters or nucleic acid sensors may compensate for reduced ABCF1 expression, potentially masking phenotypes in knockdown studies.

• Timing considerations: The kinetics of ABCF1's involvement in immune responses may be time-dependent. Perform time-course experiments to capture temporal dynamics.

• Stimulus specificity: ABCF1 responds specifically to dsDNA challenges. Ensure appropriate stimuli are used and at optimal concentrations to elicit ABCF1-dependent responses .

How does ABCF1's role as a nucleic acid sensor compare to other cytosolic DNA sensors?

ABCF1 functions as a dsDNA sensor alongside other established cytosolic DNA sensors like cGAS-STING, AIM2, and IFI16. When comparing these sensors:

• Specificity: While ABCF1 responds to dsDNA viral mimics, it appears to have broader interactions with TLR signaling pathways compared to some more specific DNA sensors .

• Downstream effects: ABCF1 primarily regulates CXCL10 and interferon-β expression, whereas other sensors may activate different inflammatory pathways or inflammasome formation.

• Cell type distribution: ABCF1 is expressed in human airway epithelial cells, making it particularly relevant for respiratory infection research .

• Evolutionary conservation: As ABC transporters are conserved across all domains of life (Archaea, Bacteria, and Eukarya), ABCF1 represents an evolutionarily ancient mechanism of nucleic acid sensing that may complement newer sensing pathways.

Understanding these distinctions can help researchers design more targeted experiments that dissect the specific contributions of ABCF1 to immune responses in different contexts.

What are the implications of ABCF1 research for understanding respiratory viral infections?

ABCF1 research has significant implications for respiratory viral infections, which are responsible for over 2.5 million deaths annually worldwide :

• Antiviral defense mechanisms: ABCF1's role in sensing viral DNA and initiating immune responses represents a critical component of the airway epithelium's defense against respiratory viruses.

• Therapeutic targets: Understanding ABCF1's function could lead to novel therapeutic approaches that enhance its activity during viral infections or compensate for deficiencies in its function.

• Chronic respiratory disease exacerbations: ABCF1 may play a role in viral-induced exacerbations of chronic respiratory diseases like asthma and COPD, offering new insights into these clinically challenging scenarios .

• Biomarker potential: Expression levels or functional variants of ABCF1 might serve as biomarkers for susceptibility to severe viral respiratory infections or poor outcomes.