ABCF3 Antibody

What is ABCF3 and why is it important in molecular research?

ABCF3 (ATP-binding cassette sub-family F member 3) is a class 2 ABC protein involved in intracellular transport processes, including the movement of molecules across cell membranes. Its dysregulation has been linked to diseases such as cancer and neurodegenerative disorders, making it a significant therapeutic target. Most notably, ABCF3 has been identified as a partner of the mouse flavivirus resistance protein 2',5'-oligoadenylate synthetase 1B (OAS1B), displaying antiviral effects against flaviviruses such as West Nile virus (WNV) . As an active ATPase whose activity is modulated by various lipids, ABCF3 presents a valuable research target for understanding cellular transport mechanisms and antiviral defense pathways.

What types of ABCF3 antibodies are currently available for research applications?

The predominant type of ABCF3 antibody available for research is rabbit polyclonal antibodies. These antibodies have been developed using various immunogens, including recombinant fusion proteins containing sequences corresponding to amino acids 1-200 of human ABCF3 (NP_060828.2) and synthetic peptides within human ABCF3 aa 650 to C-terminus . These polyclonal antibodies typically demonstrate reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species .

What are the validated applications for ABCF3 antibodies?

ABCF3 antibodies have been validated for multiple experimental techniques:

Research has confirmed positive detection in multiple tissue samples, including Jurkat cells, mouse brain, mouse lung, rat testis, and human ovary cancer tissue . For IHC applications, antigen retrieval is typically recommended with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 .

How should I validate the specificity of an ABCF3 antibody before conducting experiments?

To validate ABCF3 antibody specificity:

• Perform Western blot analysis using positive control lysates known to express ABCF3 (e.g., HeLa, 293T, or NIH 3T3 cell lysates, mouse heart tissue)

• Confirm the observed molecular weight matches the expected size (~80 kDa; though it has been observed at 85 kDa in some experiments)

• Include a negative control (e.g., IgG control for immunoprecipitation)

• Consider using tissues with differential expression of ABCF3 to confirm specificity patterns

• If possible, use ABCF3 knockdown or knockout samples as additional controls

This multi-step validation ensures your antibody specifically recognizes ABCF3 protein before proceeding with experimental applications.

How can ABCF3 antibodies be utilized to study its interaction with OAS1B in antiviral defense mechanisms?

To investigate ABCF3-OAS1B interactions in antiviral defense:

• Co-immunoprecipitation (Co-IP): Use ABCF3 antibodies to pull down protein complexes from flavivirus-infected cells, followed by Western blot analysis for OAS1B. This approach has successfully demonstrated their interaction in previous studies .

• Immunofluorescence co-localization: Apply ABCF3 antibodies in conjunction with OAS1B antibodies to visualize their co-localization at the virus-remodeled endoplasmic reticulum membrane during flavivirus infection .

• Proximity ligation assay (PLA): This technique can provide quantitative data on ABCF3-OAS1B proximity in situ, offering spatial resolution of their interaction under different conditions.

• FRET/BRET analysis: When combined with appropriately tagged constructs, these approaches can investigate the dynamics of ABCF3-OAS1B interactions in live cells during viral infection.

Recent research has established that ABCF3 significantly enhances OAS1B levels and alleviates growth inhibition caused by OAS1B expression alone. Since viral RNA synthesis requires substantial ATP, the lipid-stimulated ATP hydrolysis activity of ABCF3 may contribute to reducing viral RNA production, a characteristic feature of the flavivirus resistance phenotype .

What experimental approaches can reveal the differential roles of ABCF3's two nucleotide-binding domains?

To investigate the distinct functions of ABCF3's two nucleotide-binding domains:

• Site-directed mutagenesis: Create point mutations in each nucleotide-binding domain separately (as demonstrated in previous studies) and express these constructs in appropriate systems .

• ATPase activity assays: Compare basal and lipid-stimulated ATPase activities of wild-type ABCF3 versus domain-specific mutants. Research has shown that mutations in the two domains affect sphingosine-stimulated ATPase activity differently .

• Fluorescent ATP analog binding studies: Use fluorescence spectroscopy with ATP analogs to assess binding affinity and kinetics of each domain independently.

• Molecular dynamics simulations: Complement experimental data with computational approaches to model structural changes in each domain under different conditions.

This multi-faceted approach can support the proposed model where pocket 1 serves as the site of basal catalysis, while pocket 2 engages in ligand-stimulated ATP hydrolysis .

How can ABCF3 antibodies be employed to study the protein's modulation by different lipids?

To investigate lipid modulation of ABCF3:

• Immunoprecipitation and lipid binding assays: Use ABCF3 antibodies to isolate the protein from cellular contexts, then assess its interaction with different lipids using techniques such as lipid overlay assays or liposome binding experiments.

• Cellular fractionation combined with Western blotting: Examine ABCF3 localization to different membrane compartments under various lipid treatment conditions.

• Proximity labeling approaches: Combine ABCF3 antibodies with techniques such as APEX2 or BioID to identify proximal lipid-modifying enzymes that may influence ABCF3 function.

• Immunofluorescence co-localization with lipid probes: Visualize ABCF3 in relation to specific membrane compartments enriched in relevant lipids such as sphingomyelin, sphingosine, or cholesterol.

Research has established that ABCF3 activity is increased by sphingosine, sphingomyelin, platelet-activating factor, and lysophosphatidylcholine, while being inhibited by cholesterol. Alkyl ether lipids have shown either inhibitory effects or biphasic responses, suggesting that small changes in lipid structure can differentially affect ABCF3 activity .

What are the optimal conditions for Western blot detection of ABCF3?

For optimal Western blot detection of ABCF3:

• Sample preparation: Use whole cell lysates from appropriate positive controls such as HeLa, 293T, NIH 3T3 cells, or mouse heart tissue .

• Protein loading: Load 15-50 μg of total protein per lane for cell lysates .

• Antibody dilution: Typical working dilutions range from 1:500 to 1:2000, with 1:1000 often providing optimal results .

• Detection method: Chemiluminescence with 3-minute exposure time has been validated for detecting the approximately 80 kDa band corresponding to ABCF3 .

• Controls: Include gradient loading of positive control lysates (e.g., 5 μg, 15 μg, and 50 μg) to assess signal linearity and specificity .

Always optimize blocking conditions and washing steps for your specific experimental system to minimize background while maintaining specific signal intensity.

What are the key considerations for successful immunohistochemical detection of ABCF3?

For effective immunohistochemical detection of ABCF3:

• Tissue preparation: Formalin-fixed, paraffin-embedded tissues have been successfully used for ABCF3 detection .

• Antigen retrieval: The recommended method involves TE buffer at pH 9.0, although citrate buffer at pH 6.0 has also proven effective as an alternative .

• Antibody dilution: Start with dilutions between 1:50 and 1:500, optimizing based on your specific tissue and detection system .

• Positive control tissues: Human ovary cancer tissue and mouse heart tissue have been validated as positive controls for ABCF3 immunohistochemistry .

• Detection system: Both chromogenic and fluorescent secondary detection systems can be employed, depending on the experimental requirements.

• Counterstaining: Use appropriate nuclear counterstains that don't interfere with the evaluation of ABCF3's primarily cytoplasmic and membrane-associated localization.

Always include negative controls (omitting primary antibody) and consider dual staining with markers of subcellular compartments to better characterize ABCF3's localization patterns.

How should I optimize immunoprecipitation protocols for ABCF3 studies?

For effective immunoprecipitation of ABCF3:

• Lysate preparation: Use 1 mg of whole cell lysate (e.g., from HeLa cells) for each immunoprecipitation reaction .

• Antibody amount: 3 μg of antibody per mg of lysate has been validated for efficient ABCF3 immunoprecipitation .

• Bead selection: Protein A/G agarose or magnetic beads are suitable for pulling down rabbit IgG-ABCF3 complexes.

• Controls: Always include an isotype-matched control IgG immunoprecipitation to identify non-specific binding .

• Western blot detection: For subsequent Western blot detection of immunoprecipitated ABCF3, a dilution of 0.4 μg/ml has proven effective .

• Loading recommendation: Load approximately 20% of the immunoprecipitated material for Western blot analysis .

This protocol has been successfully employed to detect ABCF3 in immunoprecipitates, with chemiluminescent detection requiring approximately 3 minutes of exposure time .

Why might I observe ABCF3 at 85 kDa rather than the predicted 80 kDa in Western blot analysis?

The discrepancy between the calculated molecular weight (80 kDa) and the observed molecular weight (85 kDa) of ABCF3 can be attributed to several factors:

• Post-translational modifications: ABCF3 may undergo modifications such as phosphorylation, glycosylation, or ubiquitination that increase its apparent molecular weight.

• Protein structure: The tertiary structure of ABCF3 might influence its migration pattern in SDS-PAGE.

• Technical factors: Gel percentage, running buffer composition, and molecular weight markers can all affect the apparent size of proteins.

• Isoform detection: Multiple isoforms of ABCF3 may exist, with slight variations in size.

This slight discrepancy is consistent across multiple antibodies and is reported in product literature, suggesting it is an intrinsic characteristic of the protein rather than an antibody-specific artifact . When interpreting Western blot data, consider the observed molecular weight of 85 kDa as a legitimate representation of ABCF3.

How can I address non-specific binding when using ABCF3 antibodies?

To minimize non-specific binding with ABCF3 antibodies:

• Optimization of blocking: Test different blocking agents (5% non-fat dry milk, 5% BSA, or commercial blocking buffers) to identify the most effective option for your specific application.

• Antibody titration: Conduct a dilution series to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Washing optimization: Increase the number, duration, or stringency of washing steps (consider adding 0.1-0.3% Tween-20 to wash buffers).

• Pre-adsorption: For immunohistochemistry applications, consider pre-adsorbing the antibody with tissue homogenates from negative control tissues.

• Cross-reactivity assessment: Test the antibody on samples known to be negative for ABCF3 to identify potential cross-reactive proteins.

• Secondary antibody controls: Include controls omitting the primary antibody to assess non-specific binding of the secondary detection system.

Careful optimization of these parameters can significantly improve signal-to-noise ratio in ABCF3 detection assays.

How should I interpret ABCF3 expression patterns in different cellular compartments?

When analyzing ABCF3 localization patterns:

• Expectation guidance: ABCF3 has been demonstrated to localize to the endoplasmic reticulum membrane, particularly in virus-remodeled contexts when complexed with OAS1B .

• Compartment markers: Co-stain with markers for cellular compartments (e.g., calnexin for ER, GM130 for Golgi) to accurately identify ABCF3 localization.

• Context consideration: Interpret localization patterns in the context of experimental conditions, as ABCF3 distribution may change during viral infection or under different lipid environments.

• Quantitative approach: Employ quantitative image analysis to assess the relative distribution of ABCF3 across different cellular compartments.

• Validation through fractionation: Complement imaging data with biochemical fractionation and Western blot analysis to confirm subcellular distribution.

Functional interpretation should consider that as an ABC transporter family member involved in lipid-modulated ATPase activity, ABCF3's localization to specific membrane compartments likely reflects its functional roles in those locations.

How can I differentiate between specific ABCF3 signal and potential cross-reactivity with other ABC transporters?

To distinguish specific ABCF3 signal from cross-reactivity with related ABC transporters:

• Sequence comparison: Analyze the immunogen sequence used to generate the antibody for homology with other ABC transporters, particularly ABCF1 and ABCF2.

• Knockout/knockdown validation: Utilize ABCF3 knockout or knockdown samples as negative controls to confirm signal specificity.

• Peptide competition: Perform peptide competition assays using the specific immunogen peptide to verify that signal reduction occurs with the blocking peptide.

• Multiple antibody approach: Use antibodies raised against different epitopes of ABCF3 to confirm consistent detection patterns.

• Mass spectrometry validation: Following immunoprecipitation with ABCF3 antibodies, subject the pulled-down proteins to mass spectrometry analysis to confirm identity.

This multi-faceted approach ensures that the signals detected truly represent ABCF3 rather than cross-reactive ABC transporter family members.

How might ABCF3 antibodies be utilized in studying the protein's role in antiviral defense beyond flaviviruses?

To investigate ABCF3's potential role in broader antiviral defense mechanisms:

• Infection models: Apply ABCF3 antibodies in immunofluorescence and biochemical analyses of cells infected with various virus families beyond flaviviruses.

• Protein interaction networks: Use ABCF3 antibodies for immunoprecipitation followed by mass spectrometry to identify virus-specific interacting partners during different viral infections.

• ABCF3 modulation: Combine antibody-based detection methods with ABCF3 overexpression or knockdown to assess its impact on replication of diverse viruses.

• Pathway analysis: Employ ABCF3 antibodies in conjunction with markers of innate immune pathways to position ABCF3 within the broader antiviral response network.

Given ABCF3's established role in flavivirus defense through its interaction with OAS1B, exploring its potential involvement in other viral infection contexts represents a promising research direction that could reveal broader antiviral functions .

What approaches can be used to investigate potential roles of ABCF3 in neurodegenerative disorders?

To explore ABCF3's potential implications in neurodegenerative diseases:

• Clinical sample analysis: Apply ABCF3 antibodies in immunohistochemistry or Western blot analysis of post-mortem brain tissues from patients with various neurodegenerative disorders compared to healthy controls.

• Co-localization studies: Perform double immunostaining with ABCF3 antibodies and markers of disease-specific protein aggregates (e.g., tau, α-synuclein, Aβ).

• Animal model investigation: Examine ABCF3 expression patterns in established mouse models of neurodegenerative diseases using immunohistochemistry and biochemical approaches.

• Cell stress response: Investigate ABCF3 expression and localization changes in neuronal cultures subjected to stressors relevant to neurodegeneration (oxidative stress, protein misfolding, etc.).

• Genetic association studies: Correlate ABCF3 expression levels (detected via antibody-based methods) with genetic variants identified in patient populations.

Since ABCF3 dysregulation has been linked to neurodegenerative disorders, these approaches could reveal its specific contributions to disease pathogenesis or potential as a biomarker .