ABCF2 Antibody

What is ABCF2 and what cellular functions does it regulate?

ABCF2 is a member of the ATP-binding cassette (ABC) transporter superfamily, specifically belonging to subfamily F. Unlike typical ABC transporters that function in membrane transport, ABCF2 is primarily cytosolic and plays vital roles in regulating cellular functions including ribosome biogenesis and mRNA processing. The protein contributes to RNA metabolism and transport mechanisms within cells, making it an important target for investigation in various disease contexts including cancer, neurological disorders, and viral infections .

What types of ABCF2 antibodies are available for research applications?

The primary types available are polyclonal antibodies raised in rabbits that target specific epitopes of human ABCF2. These include products such as the ABCF2 Rabbit Polyclonal Antibody (CAB4365) generated against a recombinant fusion protein containing amino acids 1-250 of human ABCF2 (NP_005683.2) , and the Anti-ABCF2 Antibody (A42877) developed against a synthesized peptide derived from internal regions of human ABCF2 . Most commercially available antibodies have been validated for Western blot applications, with some also tested for immunofluorescence, immunocytochemistry, and ELISA techniques .

How do I select the appropriate ABCF2 antibody for my specific experimental needs?

When selecting an ABCF2 antibody, consider:

• Application compatibility: Verify validation data for your intended application (Western blot, immunofluorescence, etc.)

• Species reactivity: Most ABCF2 antibodies react with human samples, with some cross-reacting with mouse samples

• Epitope specificity: Consider whether the antibody targets a region of interest in ABCF2

• Validation evidence: Review published scientific validation data showing detection of endogenous ABCF2 in appropriate cell lines like HeLa, 293T, or MCF7

• Clonality requirements: For reproducibility across experiments, consider whether polyclonal variability is acceptable or if monoclonal consistency is needed

When studying specific protein domains or interactions, select antibodies targeting relevant epitopes that won't interfere with binding partners or functional regions under investigation.

What are the optimal conditions for Western blot detection of ABCF2?

For optimal Western blot detection of ABCF2:

• Sample preparation: Use cell lines with confirmed ABCF2 expression (293T, HeLa, MCF7, mouse testis, mouse brain)

• Protein amount: Load 20-50μg of total protein per lane

• Antibody dilution: Utilize a dilution range of 1:500 to 1:2000 for primary antibody

• Incubation conditions: Overnight at 4°C is recommended for primary antibody

• Detection method: HRP-conjugated secondary antibodies with ECL detection systems

• Expected molecular weight: Look for a band at approximately 71-72kDa

For challenging samples, consider increasing protein load, extending antibody incubation time, or using signal enhancement systems to improve detection sensitivity.

How can I optimize immunofluorescence protocols when using ABCF2 antibodies?

For immunofluorescence optimization with ABCF2 antibodies:

• Fixation method: 4% paraformaldehyde (10-15 minutes) works well for most applications

• Permeabilization: Use 0.2% Triton X-100 in PBS for 5-10 minutes

• Blocking: 5% normal serum (matching secondary antibody host) for 1 hour

• Primary antibody dilution: Use 1:50 to 1:200 dilution range

• Incubation time: Overnight at 4°C for maximum sensitivity

• Controls: Include:

  • Secondary-only control to assess background

  • Known positive control (e.g., HeLa cells)

  • Competitive blocking with immunizing peptide when available

For co-localization studies, ensure spectral separation between fluorophores and use sequential scanning to minimize bleed-through when performing confocal microscopy.

What validation steps should I take to confirm ABCF2 antibody specificity in my experimental system?

To validate ABCF2 antibody specificity:

• Molecular weight verification: Confirm detection at the expected 71-72kDa size

• Positive controls: Use cell lines with known ABCF2 expression (293T, HeLa, MCF7)

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR knockout of ABCF2 and confirm signal reduction

• Recombinant protein controls: If available, use purified ABCF2 protein as a positive control

• Multiple antibody comparison: Use antibodies targeting different ABCF2 epitopes and compare detection patterns

• Pre-absorption test: Pre-incubate antibody with immunizing peptide to confirm specificity

These steps collectively provide strong evidence for antibody specificity and experimental reliability across different applications.

What is the role of ABCF2 in cisplatin resistance in ovarian cancer?

ABCF2 has been identified as a critical mediator of cisplatin resistance in ovarian cancer through several mechanisms:

• NRF2 pathway regulation: ABCF2 is a direct target gene of the transcription factor NRF2, which is known to enhance chemoresistance in various cancers

• Anti-apoptotic effects: Cells with high ABCF2 expression demonstrate reduced apoptosis when exposed to cisplatin

• Functional consequences:

  • Overexpression of ABCF2 in parental ovarian cancer cells decreases cisplatin-induced apoptosis

  • Knockdown of ABCF2 in cisplatin-resistant cells increases sensitivity to cisplatin treatment

The identification of a functional antioxidant response element (ARE) in the ABCF2 promoter region establishes it as a direct NRF2 target gene, placing it within a broader network of genes involved in chemoresistance mechanisms .

How can ABCF2 expression be manipulated in experimental models to study chemoresistance?

ABCF2 expression can be experimentally manipulated through several approaches:

• Overexpression strategies:

  • Transient transfection of ABCF2 expression vectors

  • Stable cell line generation using lentiviral/retroviral systems with selection markers

  • Inducible expression systems (e.g., Tet-On/Off) for temporal control

• Knockdown/knockout approaches:

  • siRNA for transient knockdown (72-96 hour window)

  • shRNA for stable knockdown

  • CRISPR-Cas9 for complete knockout

• Pathway modulation:

  • NRF2 activators (e.g., sulforaphane) to indirectly increase ABCF2 expression

  • NRF2 inhibitors to downregulate ABCF2

Each approach should include appropriate controls and validation of ABCF2 expression levels using Western blot or qRT-PCR. For chemoresistance studies, measure cell viability, apoptosis markers, and cisplatin sensitivity using dose-response curves and IC50 determinations .

What experimental readouts should be used to assess the impact of ABCF2 on chemoresistance?

To comprehensively assess ABCF2's impact on chemoresistance, researchers should employ multiple complementary assays:

• Cell viability assays:

  • MTT/MTS/WST-1 for metabolic activity

  • Crystal violet staining for adherent cell mass

  • Real-time cell analysis systems for dynamic monitoring

• Apoptosis measurements:

  • Annexin V/PI staining and flow cytometry

  • Caspase-3/7 activity assays

  • PARP cleavage by Western blot

• Drug sensitivity parameters:

  • IC50 determination through dose-response curves

  • Colony formation assays for long-term survival

  • Drug accumulation assays to assess cellular drug uptake

• Molecular pathway analysis:

  • NRF2 pathway activation status

  • Expression of additional ABC transporters

  • Oxidative stress markers

• In vivo models (for advanced studies):

  • Xenograft models with manipulated ABCF2 expression

  • Patient-derived xenografts

  • Response to cisplatin treatment regimens

These multi-dimensional approaches provide robust evidence for ABCF2's role in chemoresistance mechanisms .

How does ABCF2 function as an adhesion receptor for Pasteurella multocida?

ABCF2 has been identified as a host cell protein that specifically mediates adherence of the zoonotic pathogen Pasteurella multocida. This function appears to be specific, as ABCF2 does not contribute to the adherence of other bacterial species such as Klebsiella pneumoniae and Bordetella bronchiseptica .

The mechanism involves:

• Specific recognition: ABCF2 appears to recognize structures on P. multocida not present on other bacterial species

• Expression regulation: P. multocida infection upregulates host ABCF2 expression through activation of p38 MAPK and NF-κB signaling pathways

• Functional impact: Overexpression of ABCF2 markedly increases bacterial adherence, while knockdown reduces it

• Downstream consequences: ABCF2 involvement in P. multocida-induced p53-dependent apoptotic signaling pathway

This represents a previously unrecognized function of ABCF2 beyond its known roles in RNA metabolism and chemoresistance.

What methodological approaches can be used to study ABCF2-pathogen interactions?

To study ABCF2-pathogen interactions, researchers can employ these methodological approaches:

• Proximity labeling techniques:

  • TurboID-based labeling to identify interacting proteins

  • BioID or APEX2 approaches for temporal interaction studies

• Infection models:

  • Cell culture infection assays with adherence/invasion quantification

  • Bacterial attachment assays with microscopy visualization

  • Host cell protein knockdown/overexpression followed by infection

• Binding studies:

  • Pull-down assays with recombinant ABCF2

  • Bacterial surface protein identification using mass spectrometry

  • Surface plasmon resonance for binding kinetics

• Signaling pathway analysis:

  • Inhibitor studies targeting p38 MAPK and NF-κB pathways

  • Phosphorylation status assessment of key signaling molecules

  • Gene expression profiling after infection

• Functional assays:

  • ABCF2 knockout/knockdown effects on bacterial adherence

  • Site-directed mutagenesis to identify critical ABCF2 domains

  • Competitive inhibition studies

These approaches provide complementary data to characterize the molecular basis of ABCF2-pathogen interactions .

How can I develop an experimental system to identify novel ABCF2 interacting partners?

For identifying novel ABCF2 interacting partners, consider these methodological approaches:

• Proximity-based labeling:

  • TurboID or BioID fusion with ABCF2 to identify proximal proteins

  • APEX2-based proximity labeling for temporal dynamics

  • These methods are particularly powerful as demonstrated in identifying ABCF2's role in P. multocida adherence

• Affinity purification coupled with mass spectrometry:

  • Tagged ABCF2 expression (FLAG, HA, GFP)

  • Gentle lysis conditions to preserve interactions

  • Differential analysis comparing bait vs. control pulldowns

  • Quantitative approaches (SILAC, TMT) for higher confidence

• Yeast two-hybrid screening:

  • Use ABCF2 domains as bait against cDNA libraries

  • Validate hits with orthogonal methods

• Protein complementation assays:

  • Split-GFP or NanoBiT systems

  • Bimolecular fluorescence complementation (BiFC)

• Crosslinking strategies:

  • Chemical crosslinking followed by MS analysis

  • Photo-crosslinking for capturing transient interactions

Each approach has strengths and limitations; combining multiple methods provides higher confidence in identifying genuine interacting partners.

What common challenges arise when working with ABCF2 antibodies and how can they be addressed?

For particularly challenging applications, consider using genetically tagged ABCF2 constructs (FLAG, HA, GFP) as alternatives to direct antibody detection.

How can ABCF2 antibodies be used in combination with other techniques for comprehensive pathway analysis?

ABCF2 antibodies can be integrated with complementary techniques for comprehensive pathway analysis:

• Chromatin immunoprecipitation (ChIP) studies:

  • Use NRF2 antibodies to confirm binding to the antioxidant response element (ARE) in the ABCF2 promoter

  • Combine with ABCF2 expression analysis to correlate transcription factor binding with expression changes

• Dual immunoprecipitation strategies:

  • Sequential IP with ABCF2 and interaction partner antibodies

  • Investigate ABCF2 role in protein complexes (e.g., ribosome-associated complexes)

• CRISPR screens with ABCF2 pathway readouts:

  • Genome-wide or targeted screens with ABCF2 expression/localization as endpoints

  • Identify regulatory factors controlling ABCF2 function

• Single-cell approaches:

  • Combine ABCF2 antibodies with other markers for mass cytometry (CyTOF)

  • Single-cell Western blot for heterogeneity analysis

• Spatial transcriptomics/proteomics:

  • Correlate ABCF2 protein localization with spatial gene expression patterns

  • Investigate microenvironmental influences on ABCF2 expression and function

• Multiomics integration:

  • Combine ABCF2 protein data with transcriptomics, metabolomics

  • Network analysis to position ABCF2 within broader cellular pathways

These integrative approaches provide deeper insights into ABCF2 biology than any single technique alone.