FAR1 Antibody, HRP conjugated

What is FAR1 protein and what cellular functions does it perform?

FAR1 (Fatty Acyl-CoA Reductase 1) is a key protein involved in cellular responses to stress and DNA damage. It plays a critical role in the reduction of fatty acids to fatty alcohols, a process essential for the synthesis of ether lipids, particularly plasmalogens. FAR1 is primarily localized to peroxisomes, where it functions as an integral membrane protein with a calculated molecular weight of approximately 59kDa (observed at 55kDa in experimental conditions) . The protein is involved in mediating cell death and survival pathways in response to external stressors, making it a significant target for investigation in cancer research. FAR1's role in regulating cell growth and DNA repair processes underscores its importance in maintaining genomic stability and preventing the development of cancerous cells .

What are the primary applications for FAR1 antibodies in research?

FAR1 antibodies serve multiple research purposes in molecular and cellular biology. They are primarily utilized in Western blot applications and ELISA assays to detect and quantify FAR1 protein expression in various cell types . Researchers employ these antibodies to investigate FAR1's involvement in DNA repair mechanisms, apoptotic pathways, and cell cycle regulation. Additionally, FAR1 antibodies have proven valuable in studying peroxisomal functions, as FAR1 is a peroxisomal protein involved in lipid metabolism. In cancer research, these antibodies help elucidate the role of FAR1 in tumor development and progression, potentially contributing to the identification of novel therapeutic targets .

What is HRP conjugation and why is it beneficial for antibody applications?

Horseradish Peroxidase (HRP) conjugation involves chemically linking the enzyme HRP to an antibody molecule, typically through cross-linking chemistry. This conjugation creates a detection system where the antibody provides specificity while the enzyme generates a detectable signal. HRP conjugation is beneficial because the enzyme catalyzes reactions with various substrates to produce colorimetric, chemiluminescent, or fluorescent signals that can be easily measured .

The basic method for conjugating HRP to antibodies involves:

• Thiolation of the antibody to introduce reactive sulfhydryl groups

• Activation of HRP with heterobifunctional cross-linkers

• Reaction of the modified antibody with activated HRP

• Purification of the resulting conjugate

This approach can be adapted for different cross-linkers depending on the specific research requirements . HRP-conjugated antibodies provide significant advantages in terms of sensitivity, stability, and versatility across various detection platforms.

How does FAR1 expression relate to plasmalogen synthesis?

FAR1 serves as a rate-limiting enzyme for plasmalogen synthesis, with direct experimental evidence showing that increased FAR1 expression enhances plasmalogen production in wild-type Chinese hamster ovary cells . Plasmalogens are a subclass of glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position, and they play crucial roles in membrane dynamics and cellular signaling.

Interestingly, a reciprocal relationship exists between FAR1 and plasmalogens – FAR1 is preferentially degraded in response to increased cellular levels of plasmalogens, indicating a negative feedback loop that maintains plasmalogen homeostasis . The region flanking the transmembrane domain of FAR1 has been identified as critical for this plasmalogen-dependent modulation of FAR1 stability. This relationship makes FAR1 antibodies essential tools for investigating the regulatory mechanisms controlling plasmalogen synthesis and the subsequent effects on cellular function .

What are the optimal conditions for using FAR1 HRP-conjugated antibodies in Western blot applications?

For optimal Western blot results with FAR1 HRP-conjugated antibodies, researchers should follow these evidence-based parameters:

When optimizing, it's crucial to consider that FAR1 appears at approximately 55kDa band in SDS-PAGE despite a calculated molecular weight of 59kDa . For studying protein interactions or modifications, less stringent lysis conditions may be required to preserve protein complexes and post-translational modifications.

How should researchers prepare samples for optimal FAR1 detection using HRP-conjugated antibodies?

Effective sample preparation is critical for successful detection of FAR1 using HRP-conjugated antibodies:

• Lysis Buffer Selection: Use buffers containing 1% NP-40 or Triton X-100 with protease inhibitors for whole-cell lysates. For peroxisomal fractions, specialized isolation protocols preserving organelle integrity are recommended.

• Protein Extraction Protocol:

  • Harvest cells at 70-80% confluence

  • Wash twice with ice-cold PBS

  • Add lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% NP-40, protease inhibitors)

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Protein Quantification: BCA or Bradford assay to ensure equal loading.

• Sample Denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer with reducing agent.

• Loading Control Selection: Use peroxisomal markers (PMP70) for subcellular specificity or housekeeping proteins (actin) for whole-cell analysis .

Since FAR1 is located in peroxisomes, subcellular fractionation methods may provide enhanced detection sensitivity by concentrating the target protein compared to whole cell lysates .

How can FAR1 antibodies be used to study the relationship between plasmalogen levels and FAR1 stability?

FAR1 antibodies provide powerful tools for investigating the unique relationship between plasmalogen levels and FAR1 stability:

• Pulse-Chase Experiments: Using FAR1 antibodies in pulse-chase assays with cells cultured under varying plasmalogen concentrations can establish degradation rates. Research has shown that FAR1, but not its homolog FAR2, undergoes preferential degradation in response to increased cellular plasmalogen levels .

• Domain Mapping: By generating constructs where regions of FAR1 are replaced with corresponding regions from FAR2 (which is not subject to plasmalogen-dependent degradation), researchers have identified that the region flanking the transmembrane domain of FAR1 is critical for this regulatory mechanism . FAR1 antibodies can detect these chimeric proteins to elucidate structural requirements.

• Subcellular Fractionation: FAR1 antibodies can be used to track the protein's localization in peroxisomal fractions under varying plasmalogen conditions, determining if degradation occurs in the peroxisome or after transport to other compartments.

• Co-immunoprecipitation: Using FAR1 antibodies for pull-down assays can identify proteins interacting with FAR1 under different plasmalogen conditions, potentially revealing the degradation machinery components.

This research direction is particularly valuable because FAR1 has been identified as a rate-limiting enzyme for plasmalogen synthesis, making the plasmalogen-dependent degradation pathway a significant regulatory mechanism in lipid metabolism .

How does phosphorylation affect FAR1 detection with antibodies?

Phosphorylation significantly impacts FAR1 detection with antibodies through several mechanisms:

• Mobility Shifts: Phosphorylated forms of FAR1 migrate slower on SDS-PAGE gels, appearing as multiple bands with higher apparent molecular weights. These modified species can be confirmed as phosphorylated forms through treatment with alkaline phosphatase, which collapses the multiple bands to a single band .

• Epitope Masking: Phosphorylation can alter protein conformation and potentially mask antibody binding sites. This is particularly relevant for FAR1, where serine 87 phosphorylation is known to trigger degradation . Antibodies targeting regions containing or adjacent to phosphorylation sites may show differential binding depending on phosphorylation status.

• Stability Effects: Phosphorylation of FAR1 at serine 87 promotes its degradation, which directly affects protein abundance and detection sensitivity . The mutation of serine 87 to proline (as in the Far1-22 mutant) stabilizes the protein by preventing phosphorylation-dependent degradation.

• Subcellular Localization: Phosphorylation may influence FAR1's subcellular distribution between nuclear and cytoplasmic compartments, which in turn affects degradation rates . Research has shown that cytoplasmic FAR1 is more stable than nuclear FAR1, suggesting compartment-specific regulation.

For comprehensive analysis, researchers should consider using phospho-specific antibodies alongside total FAR1 antibodies to distinguish between different phosphorylated forms and their functional implications .

What are common issues with FAR1 HRP-conjugated antibodies and how can they be resolved?

Research has shown that FAR1 detection can be particularly challenging due to its regulation by plasmalogens and its differential stability based on subcellular localization. Nuclear FAR1 is degraded more rapidly than cytoplasmic FAR1, which may affect detection consistency . Additionally, FAR1's calculated molecular weight (59kDa) differs from its observed migration on SDS-PAGE (55kDa), which should be considered when interpreting Western blot results .

How can researchers validate the specificity of FAR1 HRP-conjugated antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For FAR1 HRP-conjugated antibodies, implement these approaches:

• Positive and Negative Controls:

  • Use cell lines known to express FAR1 (Daudi, HepG2) as positive controls

  • Include negative controls as specified in the antibody datasheet

  • Compare expression patterns with published literature

• Genetic Validation:

  • Test antibody in FAR1 knockout or knockdown models

  • Use siRNA/shRNA against FAR1 to create expression gradients

  • Overexpress FAR1 to confirm increased signal intensity

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Specific binding should be blocked while non-specific binding persists

• Cross-Reactivity Assessment:

  • Test against recombinant FAR1 and the homologous FAR2 protein

  • Evaluate using chimeric constructs containing regions of FAR1 and FAR2

• Orthogonal Detection Methods:

  • Confirm results using antibodies targeting different FAR1 epitopes

  • Validate with mass spectrometry identification of immunoprecipitated proteins

• Molecular Weight Verification:

  • Confirm detection at the expected molecular weight (55kDa observed, 59kDa calculated)

  • Account for post-translational modifications that may alter migration patterns

Researchers should document validation results thoroughly, as antibody specificity can vary between applications and experimental conditions.

How can FAR1 antibodies contribute to cancer research and potential therapeutic developments?

FAR1 antibodies represent valuable tools in cancer research through several important mechanisms:

• Biomarker Identification: FAR1 is involved in mediating cell death and survival pathways in response to external stressors, making it a potential biomarker for cancer progression and treatment response . HRP-conjugated antibodies enable sensitive detection in tissue microarrays and patient samples.

• DNA Repair Pathway Analysis: FAR1's involvement in DNA repair processes makes it relevant to understanding mechanisms of resistance to genotoxic therapies . Antibodies can help map interaction networks between FAR1 and other repair proteins through co-immunoprecipitation studies.

• Cellular Stress Response Monitoring: Using FAR1 antibodies, researchers can track how cancer cells modulate FAR1 expression and localization in response to therapeutic interventions that induce cellular stress.

• Lipid Metabolism Alterations: Since FAR1 is a rate-limiting enzyme in plasmalogen synthesis , and cancer cells often exhibit altered lipid metabolism, FAR1 antibodies can help characterize metabolic reprogramming in tumors.

• Target Validation: For potential therapeutic approaches targeting FAR1 or its regulatory pathways, antibodies provide essential tools for confirming target engagement and pathway modulation.

• Personalized Medicine Applications: Quantitative assessment of FAR1 expression in patient samples using calibrated antibody assays could potentially guide treatment selection for individual patients.

As research continues to elucidate FAR1's role in maintaining genomic stability and preventing cancer development , antibodies with optimized sensitivity and specificity will remain instrumental in translating these findings toward clinical applications.