FARSB Antibody, Biotin conjugated

What is FARSB and why is it studied in research?

FARSB (Phenylalanine-tRNA Synthetase Beta Subunit) is a critical component of the phenylalanine-tRNA synthetase complex that catalyzes the attachment of phenylalanine to its cognate tRNA during protein synthesis. This 66 kDa protein (Q9NSD9) functions in the cytoplasm and is essential for accurate translation of the genetic code . Research interest in FARSB stems from its fundamental role in protein synthesis and its implications in various cellular processes. The study of FARSB through antibody-based approaches allows researchers to investigate its expression, localization, and functional interactions in diverse physiological and pathological contexts .

What are the key specifications of commercially available FARSB antibodies with biotin conjugation?

The FARSB antibody with biotin conjugation is typically a rabbit polyclonal antibody with confirmed reactivity to human, mouse, and rat FARSB proteins . The antibody has a predicted molecular weight of 66 kDa and has been validated for Western blot (WB) and immunohistochemistry (IHC) applications . Some preparations have been assigned the Research Resource Identifier (RRID) AB_2845963 for consistent reference in published research . Biotin conjugation enables versatile detection strategies using avidin/streptavidin systems, enhancing sensitivity in various experimental contexts .

How does biotin conjugation enhance antibody functionality in FARSB detection?

Biotin conjugation creates a versatile detection platform through the exploitation of the high-affinity interaction between biotin and avidin/streptavidin (Kd ≈ 10^-15 M) . This conjugation allows for signal amplification through multiple binding sites on avidin/streptavidin molecules and permits various detection strategies using fluorescent, enzymatic, or particulate labels . For FARSB antibodies specifically, biotin conjugation facilitates sensitive detection in applications like immunohistochemistry and Western blotting without affecting the antibody's binding specificity to the FARSB target . The biotin-XX conjugates, which contain an extended spacer arm, minimize steric hindrance and maintain high avidin/streptavidin binding affinity even when the biotin is attached to the antibody .

What are the optimal dilution conditions for FARSB antibody (biotin conjugated) in Western blot applications?

While optimal dilutions should be determined empirically for each specific application and experimental condition, biotin-conjugated FARSB antibodies typically perform well in Western blot applications at dilutions ranging from 1:500 to 1:2000 . For optimal results, researchers should perform a titration experiment using different dilutions (e.g., 1:500, 1:1000, 1:2000) against a positive control sample known to express FARSB. Blocking should be performed with BSA rather than milk proteins when using biotin-conjugated antibodies to avoid interference from endogenous biotin in milk . Detection can be achieved using streptavidin-HRP conjugates followed by enhanced chemiluminescence (ECL) visualization .

How can biotin-conjugated FARSB antibodies be effectively employed in immunohistochemistry?

For effective immunohistochemical detection of FARSB using biotin-conjugated antibodies, follow this optimized protocol:

• Deparaffinize and rehydrate tissue sections using standard procedures

• Perform antigen retrieval (typically heat-induced epitope retrieval at pH 6.0)

• Block endogenous biotin using a commercial biotin blocking kit to reduce background

• Apply biotin-conjugated FARSB antibody (typically at 1:100 to 1:500 dilution) and incubate at 4°C overnight

• Wash thoroughly with PBS containing 0.05% Tween-20

• Apply streptavidin-conjugated detection reagent (HRP, alkaline phosphatase, or fluorophore)

• Develop signal using appropriate substrate or visualize with fluorescence microscopy

For maximal sensitivity, researchers can implement tyramide signal amplification (TSA) or enzyme-labeled fluorescence (ELF) techniques as described in the literature .

What detection systems work best with biotin-conjugated FARSB antibodies?

The most effective detection systems for biotin-conjugated FARSB antibodies leverage the strong biotin-avidin/streptavidin interaction. Streptavidin conjugates generally provide lower background than avidin conjugates due to reduced non-specific binding . The following detection systems have demonstrated excellent performance:

For maximal sensitivity, tyramide signal amplification techniques using streptavidin-HRP can increase detection by 10-100 fold compared to conventional methods .

How can background issues be minimized when using biotin-conjugated FARSB antibodies?

Background issues with biotin-conjugated antibodies often result from endogenous biotin in tissues or non-specific binding. Implement these strategies to minimize background:

• Use a commercial biotin blocking kit before antibody application to block endogenous biotin

• Replace avidin with streptavidin or NeutrAvidin conjugates, which exhibit less non-specific binding

• Increase washing steps duration and stringency (use 0.1-0.3% Triton X-100 in wash buffers)

• Optimize antibody dilution - excess antibody contributes to background

• Use BSA instead of milk for blocking and antibody dilution buffers

• For tissues with high endogenous biotin (kidney, liver), consider alternative detection methods or implement specific blocking steps with free unconjugated biotin and avidin

For particularly problematic samples, adding free D-biotin (10 mM) after the streptavidin-fluorophore incubation can enhance signal-to-noise ratio by reversing some non-specific interactions .

What factors affect the stability and shelf-life of biotin-conjugated FARSB antibodies?

Biotin-conjugated FARSB antibodies have specific storage requirements to maintain functionality. Key factors affecting stability include:

• Storage temperature: Optimal at -20°C for long-term storage; 4°C for short-term (≤1 month)

• Freeze-thaw cycles: Minimize cycles; aliquot upon receipt to prevent degradation

• Buffer composition: Presence of preservatives (0.02-0.05% sodium azide) enhances stability

• Light exposure: Protect from light, especially if conjugated with fluorophores

• Protein concentration: Higher concentrations generally provide better stability

• Contamination: Use sterile techniques when handling

The typical shelf-life of properly stored biotin-conjugated antibodies is 12-18 months, though activity should be verified before use in critical experiments . Degradation often manifests as reduced signal intensity and increased background in applications.

How can researchers validate the specificity of biotin-conjugated FARSB antibodies?

Validating specificity is crucial for ensuring reliable experimental results. Implement these validation approaches:

• Positive and negative control samples: Use tissues/cells known to express or lack FARSB

• Western blot analysis: Confirm detection of a single band at the expected molecular weight (66 kDa)

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

• Knockdown/knockout validation: Compare staining between wild-type and FARSB-depleted samples

• Cross-validation with multiple antibodies: Compare staining patterns with other validated FARSB antibodies

• Immunoprecipitation followed by mass spectrometry: Confirm that the precipitated protein is indeed FARSB

For biotin-conjugated antibodies specifically, also test with streptavidin alone (no primary antibody) to assess background from endogenous biotin or non-specific streptavidin binding .

How can biotin-conjugated FARSB antibodies be utilized in multiplex immunostaining protocols?

Multiplex immunostaining allows simultaneous detection of multiple proteins in a single sample. For incorporating biotin-conjugated FARSB antibodies in multiplex protocols:

• Sequential detection approach:

  • Apply unconjugated primary antibodies from different species

  • Detect with species-specific secondary antibodies conjugated to different fluorophores

  • Block with excess unconjugated secondary antibody

  • Apply biotin-conjugated FARSB antibody

  • Detect with a spectrally distinct fluorophore-conjugated streptavidin

• Tyramide signal amplification (TSA) approach:

  • Perform sequential TSA labeling with heat-mediated antibody stripping between rounds

  • Include biotin-conjugated FARSB antibody in one round

  • This approach permits use of antibodies from the same species without cross-reactivity

• Consider using CMNB-caged fluorescein conjugates of streptavidin for photoactivated fluorescence, which allows precise spatial control of detection .

What are the considerations for using biotin-conjugated FARSB antibodies in affinity purification experiments?

Biotin-conjugated FARSB antibodies can be powerful tools for affinity purification of FARSB and its binding partners. Key considerations include:

• Immobilization strategy:

  • Use streptavidin-agarose or streptavidin-magnetic beads as the solid support

  • Saturate binding capacity with biotin-conjugated antibody

  • Cross-link antibody to streptavidin if harsh elution conditions will be used

• Binding conditions:

  • Optimize salt concentration, pH, and detergent type/concentration

  • Include protease inhibitors and phosphatase inhibitors if studying post-translational modifications

  • Pre-clear lysates to reduce non-specific binding

• Elution strategies:

  • Competitive elution with biotin is not recommended as it disrupts the antibody-streptavidin interaction

  • Use low pH, high salt, or denaturing conditions for elution

  • For gentler elution, consider antibodies conjugated with DSB-X biotin, which allows reversible binding

For co-immunoprecipitation studies identifying FARSB interaction partners, use physiological buffers with mild detergents (0.1% NP-40 or 0.1% Triton X-100).

How can biotin-conjugated FARSB antibodies be employed in studying protein-RNA interactions?

Given FARSB's role in tRNA binding, biotin-conjugated FARSB antibodies can provide valuable insights into protein-RNA interactions through these approaches:

• RNA immunoprecipitation (RIP):

  • Cross-link protein-RNA complexes in vivo using formaldehyde or UV

  • Immunoprecipitate using biotin-conjugated FARSB antibody and streptavidin beads

  • Extract and analyze associated RNAs through RT-PCR or sequencing

• Proximity ligation assay (PLA) for RNA visualization:

  • Use biotin-conjugated FARSB antibody and a second antibody against a suspected interacting protein

  • Apply oligonucleotide-conjugated streptavidin and species-specific secondary antibody

  • Perform rolling circle amplification and hybridize with fluorescent probes

• CLIP-seq (Cross-linking immunoprecipitation followed by sequencing):

  • UV cross-link protein-RNA complexes in vivo

  • Immunoprecipitate using biotin-conjugated FARSB antibody

  • Process samples for high-throughput sequencing to map RNA binding sites

These approaches can reveal the RNA binding specificity and dynamics of FARSB in different cellular contexts, providing insights into its function beyond canonical tRNA charging activities.

What are the technical considerations for using biotin-conjugated FARSB antibodies in super-resolution microscopy?

Super-resolution microscopy techniques can resolve structures below the diffraction limit, providing detailed insights into FARSB localization. When using biotin-conjugated FARSB antibodies for these applications:

• Detection system selection:

  • Use small fluorophore-conjugated streptavidin (Alexa Fluor 647 or similar) for STORM/PALM

  • For STED microscopy, select STED-compatible fluorophores conjugated to streptavidin

  • Consider using fluorophore-quencher pairs for FRET-based approaches

• Sample preparation:

  • Use thinner sections (≤5 μm) for better resolution

  • Optimize fixation to preserve structural details while maintaining epitope accessibility

  • Consider using expansion microscopy protocols for physical sample enlargement

• Signal enhancement:

  • For single-molecule localization, ensure appropriate fluorophore density

  • The biotin-streptavidin system provides natural signal amplification

  • Consider adding free biotin (B-1595, B-20656) as a final step to enhance fluorescence of some streptavidin conjugates

• Controls:

  • Include samples stained with unconjugated primary antibody plus biotinylated secondary and fluorophore-streptavidin for comparison

  • Verify specificity using knockout/knockdown controls at super-resolution level

Super-resolution imaging with biotin-conjugated FARSB antibodies can reveal previously undetectable details about FARSB's subcellular organization and potential colocalization with tRNAs or translation machinery components.

How can biotin-conjugated FARSB antibodies be integrated into high-throughput screening approaches?

Integration of biotin-conjugated FARSB antibodies into high-throughput screening offers powerful approaches for drug discovery and functional genomics:

• Automated immunofluorescence in cell microarrays:

  • Utilize biotin-conjugated FARSB antibodies for detection in cellular screens

  • Combine with streptavidin-fluorophore conjugates for rapid automated imaging

  • Quantify signal intensity changes in response to genetic or chemical perturbations

• Bead-based multiplex assays:

  • Couple different capture antibodies to spectrally distinct beads

  • Use biotin-conjugated FARSB antibody and fluorescent streptavidin for detection

  • Analyze by flow cytometry for quantitative assessment across multiple conditions

• High-content screening:

  • Implement in plate-based format for simultaneous assessment of multiple parameters

  • Monitor FARSB levels, localization, and post-translational modifications

  • Combine with markers for cellular stress, translation rate, or tRNA charging status

These approaches enable systematic investigation of compounds or genetic factors that influence FARSB function, potentially revealing new regulatory mechanisms or therapeutic targets.

What methodological approaches can enhance reproducibility when working with biotin-conjugated FARSB antibodies?

Achieving reproducible results with biotin-conjugated antibodies requires rigorous methodological considerations:

• Standardization practices:

  • Use consistent lot numbers when possible, or validate new lots against previous ones

  • Include positive control samples in each experimental run

  • Document detailed protocols including incubation times, temperatures, and buffer compositions

• Quantification approaches:

  • Implement quantitative image analysis with appropriate controls

  • Use calibration standards for fluorescence or enzymatic readouts

  • Account for non-linear signal response in highly sensitive detection systems

• Critical validation steps:

  • Verify antibody specificity through knockout/knockdown controls

  • Test for potential interference from endogenous biotin in experimental systems

  • Compare results between biotin-conjugated and unconjugated antibody formats

• Metadata reporting:

  • Document RRID number (AB_2845963) in publications

  • Report detailed antibody information including clonality, host species, supplier, and catalog number

  • Share raw data and analysis code when possible