FRMD5 Antibody, Biotin conjugated

What is FRMD5 and why is it studied in research?

FRMD5 (FERM Domain Containing 5) is a protein encoded by the FRMD5 gene located at chromosomal position 15q15.3 in humans. It is a member of the FERM domain-containing protein family, which plays crucial roles in cytoskeletal organization, membrane protein linkage, and signal transduction pathways. The FERM (4.1 protein, Ezrin, Radixin, Moesin) domain is a structural module involved in localizing proteins to the plasma membrane. Research on FRMD5 is important for understanding cytoskeletal dynamics, cell adhesion, and potential involvement in various cellular processes and disease mechanisms .

What are the key characteristics of biotin-conjugated FRMD5 antibodies?

Biotin-conjugated FRMD5 antibodies are immunological tools where the antibody targeting FRMD5 has been chemically linked to biotin molecules. These antibodies are typically polyclonal, raised in rabbits, and purified through Protein G or Protein A affinity chromatography with purity levels exceeding 95% . They target specific amino acid sequences within the FRMD5 protein, with common targeting regions including AA 211-312 and AA 295-504 . The biotin conjugation enables strong binding to streptavidin or avidin, making these antibodies valuable for detection systems in various immunoassay applications requiring signal amplification and high sensitivity .

What applications are biotin-conjugated FRMD5 antibodies suitable for?

Biotin-conjugated FRMD5 antibodies are suitable for multiple research applications, particularly those benefiting from the biotin-streptavidin detection system. Primary applications include ELISA (Enzyme-Linked Immunosorbent Assay), which is the most commonly validated application across multiple products . Additionally, these antibodies can be used in immunohistochemistry on both frozen (IHC-fro) and paraffin-embedded sections (IHC-p), providing visualization of FRMD5 distribution in tissue samples . Western blotting applications are also documented for certain biotin-conjugated FRMD5 antibodies, allowing for protein detection following electrophoretic separation. The biotin conjugation provides flexibility for various detection systems across these applications .

How should researchers select the appropriate FRMD5 antibody epitope region for their experiments?

When selecting an FRMD5 antibody, researchers should consider the specific epitope region based on their experimental needs. Available antibodies target different amino acid sequences including AA 75-124, AA 211-312, and AA 295-504 . For studying full-length FRMD5 protein, antibodies targeting the 295-504 region may provide better recognition of the native protein structure. For experiments investigating specific domains or protein interactions, researchers should select antibodies targeting the relevant functional domain. Additionally, researchers should consider cross-reactivity requirements; if cross-species studies are planned, antibodies with predicted reactivity across multiple species (e.g., mouse, rat, cow) would be preferable . Epitope mapping data and immunogen information should be carefully reviewed to ensure the antibody will detect the FRMD5 region of interest in your experimental system.

What controls should be included when using biotin-conjugated FRMD5 antibodies?

Proper experimental controls are essential when using biotin-conjugated FRMD5 antibodies. Researchers should include:

• Positive controls: Tissues or cell lines known to express FRMD5 (e.g., human cell lines with validated FRMD5 expression).

• Negative controls:

  • Isotype controls: Biotin-conjugated rabbit IgG lacking FRMD5 specificity

  • Secondary-only controls: Omitting the primary antibody to assess non-specific binding

  • Tissues or cells lacking FRMD5 expression

• Blocking controls: Pre-incubation with endogenous biotin blocking reagents to prevent non-specific binding to endogenous biotin in samples

• Peptide competition controls: Pre-incubation of the antibody with the immunizing peptide (e.g., recombinant human FRMD5 protein fragments) to confirm binding specificity

These controls help distinguish specific from non-specific signals and validate experimental findings, particularly important given the potential for endogenous biotin interference in certain tissues and the polyclonal nature of the available antibodies .

What are the optimal storage and handling conditions for biotin-conjugated FRMD5 antibodies?

Biotin-conjugated FRMD5 antibodies require specific storage and handling conditions to maintain their functionality. Upon receipt, antibodies should be aliquoted to avoid repeated freeze-thaw cycles, which can degrade the antibody and reduce binding efficiency . Storage recommendations include:

• Shipping temperature: Typically shipped at 4°C

• Long-term storage: -20°C to -80°C for maximum stability

• Storage buffer: Typically formulated in buffers containing:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 or other preservatives

For handling, researchers should:

• Avoid repeated freeze-thaw cycles

• Briefly centrifuge vials after thawing to collect contents

• Handle with gloves and appropriate PPE

• Protect biotin-conjugated antibodies from prolonged exposure to light

• Allow solutions to reach room temperature before opening vials to prevent condensation

Following these storage and handling guidelines will help maintain antibody integrity and experimental reproducibility over time.

What are the recommended dilution factors for different applications of biotin-conjugated FRMD5 antibodies?

Optimal dilution factors for biotin-conjugated FRMD5 antibodies vary by application and should be empirically determined for each specific experiment. Based on available product documentation, the following ranges serve as starting points:

Researchers should perform initial titration experiments to determine the optimal concentration that provides maximum specific signal with minimal background . The polyclonal nature of these antibodies may result in batch-to-batch variation, necessitating optimization with each new lot.

How can researchers optimize signal-to-noise ratio when using biotin-conjugated FRMD5 antibodies?

Optimizing signal-to-noise ratio is crucial for accurate FRMD5 detection using biotin-conjugated antibodies. Several methodological approaches can enhance specificity:

• Endogenous biotin blocking: Pre-incubate samples with avidin/streptavidin followed by free biotin to block endogenous biotin, which is particularly important in biotin-rich tissues (e.g., liver, kidney).

• Blocking optimization: Use 5-10% normal serum from the species of the secondary reagent, with addition of 0.1-0.3% Triton X-100 for permeabilization in immunohistochemistry applications.

• Antibody titration: Perform detailed dilution series to identify the concentration that maximizes specific signal while minimizing background.

• Incubation conditions:

  • Temperature: 4°C for longer incubations (overnight) to enhance specificity

  • Duration: Optimize between 1-24 hours depending on application

  • Washing: Multiple (3-5) extended washes with PBS-T (0.05% Tween-20)

• Detection system selection: Utilize high-sensitivity detection systems compatible with biotin, such as:

  • ABC (Avidin-Biotin Complex) systems

  • Streptavidin-HRP conjugates

  • Tyramide signal amplification for low-abundance targets

These optimizations should be systematically evaluated and documented to establish reproducible protocols for FRMD5 detection.

What is the expected molecular weight of FRMD5 in Western blot applications?

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications may increase the apparent molecular weight.

• Protein isoforms: Alternative splicing may generate multiple isoforms with different molecular weights.

• Protein degradation: Sample preparation conditions may result in degradation products appearing as lower molecular weight bands.

• Cross-reactivity: The polyclonal nature of available antibodies may result in recognition of related proteins.

Researchers should validate their results by comparing with positive controls and published literature. Antibodies targeting different epitopes (AA 211-312 vs. AA 295-504) may show slight variations in detection patterns . A comprehensive Western blot optimization should include gradient gels (8-12%) to achieve optimal separation in the 60-70 kDa range for accurate FRMD5 detection.

How can biotin-conjugated FRMD5 antibodies be utilized in multi-color immunofluorescence studies?

Biotin-conjugated FRMD5 antibodies offer significant advantages in multi-color immunofluorescence studies due to their compatibility with diverse detection systems. For optimal multi-color experiments:

• Detection strategy: Utilize streptavidin conjugated to different fluorophores (e.g., Alexa Fluor 488, 555, 594, 647) for flexible integration with other primary antibodies. Available excitation/emission profiles include:

  • AF350: 346nm/442nm

  • AF488: 493nm/519nm

  • AF555: 555nm/565nm

  • AF594: 591nm/614nm

  • AF647: 651nm/667nm

  • AF680: 679nm/702nm

  • AF750: 749nm/775nm

• Sequential staining approach: To prevent cross-reactivity:

  • Apply and detect FRMD5 antibody first

  • Block remaining biotin binding sites with excess free biotin

  • Proceed with additional primary antibodies

  • Use directly conjugated secondary antibodies for other targets

• Spectral unmixing: For advanced confocal microscopy applications, employ spectral unmixing algorithms to distinguish overlapping fluorescent signals.

• Counterstaining compatibility: DAPI nuclear counterstain (Ex/Em: 358/461 nm) presents minimal spectral overlap with common streptavidin-fluorophore conjugates used for FRMD5 detection .

This approach enables co-localization studies of FRMD5 with other proteins of interest, providing insights into protein interactions and cellular distribution patterns.

What are the considerations for using biotin-conjugated FRMD5 antibodies in proximity ligation assays (PLA)?

Proximity Ligation Assay (PLA) is an advanced technique for detecting protein-protein interactions in situ, and biotin-conjugated FRMD5 antibodies can be adapted for this application with careful methodological considerations:

• Antibody pairing strategy: For FRMD5 interaction studies, pair the biotin-conjugated FRMD5 antibody with a non-biotinylated antibody against the potential interaction partner. Ensure both antibodies are:

  • Raised in different host species

  • Validated for immunofluorescence applications

  • Capable of recognizing native protein conformations

• Secondary probe selection: Use PLA probes that recognize:

  • Anti-biotin antibody or streptavidin-conjugated PLA probe for FRMD5 detection

  • Species-specific antibody for the interaction partner

• Validation controls:

  • Positive control: Known protein interactions

  • Negative control: Proteins known not to interact with FRMD5

  • Technical controls: Single antibody controls to assess background signal

• Signal optimization:

  • Pre-block endogenous biotin

  • Optimize antibody dilutions (typically 1:100 - 1:500)

  • Increase stringency of washes to reduce non-specific interactions

• Analysis considerations:

  • Quantify PLA signals per cell

  • Compare signal distribution with conventional co-localization studies

  • Correlate with functional assays to validate interactions

This approach enables detection of native FRMD5 protein interactions within the cellular context, potentially revealing novel binding partners and functional relationships.

How can researchers troubleshoot non-specific binding when using biotin-conjugated FRMD5 antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. For FRMD5 detection, researchers can implement the following troubleshooting strategies:

• Endogenous biotin interference:

  • Problem: Many tissues contain endogenous biotin that can cause background

  • Solution: Implement avidin/biotin blocking steps before antibody application

  • Validation: Include a no-primary antibody control treated with blocking reagents

• Cross-reactivity with other FERM domain proteins:

  • Problem: The polyclonal nature of available antibodies may recognize conserved domains

  • Solution: Pre-absorb antibody with recombinant proteins containing similar FERM domains

  • Validation: Compare staining patterns with antibodies targeting different FRMD5 epitopes

• Fixation artifacts:

  • Problem: Overfixation can mask epitopes or create artificial binding sites

  • Solution: Optimize fixation conditions (4% PFA for 10-15 minutes) and implement appropriate antigen retrieval methods

  • Validation: Compare different fixation protocols and retrieval techniques

• Sample-specific background:

  • Problem: Certain tissues may exhibit high background

  • Solution: Implement longer blocking steps (2+ hours) with 5-10% normal serum and 1% BSA

  • Validation: Include isotype controls at equivalent concentrations

• Detection system optimization:

  • Problem: Signal amplification can increase background

  • Solution: Titrate streptavidin conjugates and reduce incubation times

  • Validation: Compare different detection methods to identify optimal signal-to-noise ratio

Systematic evaluation and documentation of these parameters will help establish reliable protocols for specific FRMD5 detection across experimental systems.

What is known about FRMD5 expression patterns across different tissues?

Current research on FRMD5 expression patterns remains limited, making immunodetection studies with well-validated antibodies particularly valuable. Based on available data:

Researchers investigating FRMD5 expression should implement comprehensive validation using multiple detection methods, as expression levels may vary across developmental stages and pathological conditions. The polyclonal antibodies targeting different epitope regions (AA 211-312 versus AA 295-504) may reveal distinct expression patterns, potentially indicating tissue-specific isoforms or post-translational modifications .

How can researchers validate FRMD5 antibody specificity in their experimental system?

Rigorous validation of FRMD5 antibody specificity is essential for generating reliable research data. A comprehensive validation approach should include:

• Genetic validation:

  • FRMD5 knockdown/knockout: Compare antibody staining between wild-type and FRMD5-depleted samples

  • Overexpression: Detect increased signal in FRMD5-overexpressing systems

• Biochemical validation:

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

  • Immunoprecipitation-mass spectrometry: Confirm target protein identity

  • Sequential immunoprecipitation: Deplete FRMD5 and confirm antibody specificity

• Cross-platform validation:

  • Correlate protein detection with mRNA expression

  • Compare staining patterns across multiple antibodies targeting different FRMD5 epitopes

  • Validate across different detection methods (Western blot, IHC, IF)

• Cross-reactivity assessment:

  • Test on tissues from different species when using antibodies with predicted cross-reactivity

  • Include related FERM domain proteins as specificity controls

Documentation of these validation steps is critical for publication and should be included in research methods sections to support experimental findings.

What emerging applications exist for studying FRMD5 in disease research?

While FRMD5 remains relatively understudied, emerging research suggests potential roles in various disease processes, creating opportunities for applying biotin-conjugated FRMD5 antibodies in novel contexts:

• Cardiovascular research:

  • FRMD5 may play roles in cardiac development and function

  • Potential applications in cardiomyocyte organization and cellular architecture studies

  • Investigation of cytoskeletal dynamics in cardiac pathologies

• Neurological disorders:

  • FERM domain proteins contribute to neuronal development and synaptic organization

  • Opportunities for studying FRMD5 in neurodevelopmental disorders

  • Potential roles in neuronal cytoskeletal dynamics

• Cancer biology:

  • Investigation of FRMD5 in cell migration and invasion

  • Potential roles in epithelial-mesenchymal transition

  • Opportunities for biomarker development using tissue microarrays

• Immunopathology:

  • Studying FRMD5 in immune cell function and migration

  • Potential involvement in inflammatory signaling pathways

For these emerging applications, biotin-conjugated FRMD5 antibodies offer advantages including signal amplification capabilities and compatibility with automated staining platforms for high-throughput studies . Researchers exploring these new directions should implement rigorous validation protocols and consider multiple detection methodologies to generate robust findings.