FRMD5 Antibody

What is FRMD5 and what cellular functions is it associated with?

FRMD5 (FERM domain-containing protein 5) is a 570 amino acid single-pass membrane protein containing one FERM domain. The protein exists in up to two alternatively spliced isoforms and is primarily localized to the cell membrane. FRMD5 is suspected to be involved in the regulation of cell migration. Like other proteins containing FERM domains, it likely plays a role in linking cytoskeletal signals to membrane dynamics, which is crucial for cellular processes such as adhesion, migration, and maintaining structural integrity. The FERM domain is roughly 150 amino acids in length and is characteristically found in cytoskeletal-associated proteins including Ezrin, Radixin, Moesin, and protein 4.1 .

What species reactivity can be expected with commercially available FRMD5 antibodies?

Most commercially available FRMD5 antibodies demonstrate reactivity with human samples. Many also cross-react with mouse and rat proteins. Some antibodies offer broader reactivity profiles, detecting FRMD5 in species such as cow, dog, guinea pig, horse, rabbit, and zebrafish. When selecting an antibody for your research, it's important to verify the specific reactivity profile for your species of interest. The predicted reactivity percentages can vary significantly between species, with some antibodies showing 100% reactivity in human, mouse, rat, cow, and dog samples, but reduced reactivity (e.g., 93% in rabbit, 86% in zebrafish) in other species .

What are the key distinctions between different types of FRMD5 antibodies available for research?

FRMD5 antibodies vary in several key characteristics that affect their experimental utility:

For rigorous experimental design, researchers should select antibodies validated for their specific application and with binding specificity to the region of interest in their experimental system .

What are the optimal application conditions for using FRMD5 antibodies in Western blotting?

For optimal Western blotting results with FRMD5 antibodies, follow these methodological guidelines:

• Sample preparation: Use RIPA or NP-40 buffer supplemented with protease inhibitors for cell lysis

• Loading: 20-50 μg of total protein per lane is typically sufficient for detection

• Separation: 8-10% SDS-PAGE gels provide optimal resolution for the 65.1 kDa FRMD5 protein

• Transfer: Semi-dry or wet transfer to PVDF membranes (0.45 μm pore size) is recommended

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute according to manufacturer recommendations (typically 1:300-1:5000) and incubate overnight at 4°C

• Detection: Both chemiluminescence (for HRP-conjugated systems) and fluorescence-based detection methods are compatible

When probing for FRMD5, researchers should be aware that the canonical protein has a reported mass of 65.1 kDa, but alternative splice variants may appear at different molecular weights .

How should researchers optimize immunofluorescence protocols for FRMD5 detection in different tissue types?

Immunofluorescence detection of FRMD5 requires protocol optimization based on tissue type and fixation method:

• Fixation optimization:

  • Cell cultures: 4% paraformaldehyde (10-15 minutes) preserves membrane protein structure

  • Frozen tissues: 4% paraformaldehyde followed by sucrose cryoprotection

  • Paraffin-embedded tissues: Heat-induced epitope retrieval (citrate buffer, pH 6.0) is critical

• Permeabilization: Use 0.1-0.3% Triton X-100 carefully, as excessive permeabilization may disrupt membrane proteins

• Antibody dilution: For most FRMD5 antibodies, a 1:50-1:200 dilution range is recommended for IF applications

• Counterstaining considerations: When studying FRMD5 subcellular localization, combine with membrane markers (e.g., Na+/K+ ATPase) for co-localization studies

• Controls: Include both positive controls (tissues known to express FRMD5) and negative controls (primary antibody omission)

For optimal results, researchers should be aware that FRMD5's membrane localization may require careful optimization of permeabilization steps to balance accessibility and preservation of structure .

What validation techniques should be employed to confirm FRMD5 antibody specificity?

Comprehensive validation of FRMD5 antibody specificity should include multiple complementary approaches:

• Western blot validation:

  • Positive controls: Lysates from tissues/cells known to express FRMD5

  • Negative controls: FRMD5 knockout/knockdown samples

  • Expected single band at approximately 65.1 kDa (canonical isoform)

• Immunoprecipitation followed by mass spectrometry:

  • Confirms antibody captures intended target

  • Identifies potential cross-reacting proteins

• Peptide competition assays:

  • Pre-incubation with immunizing peptide should abolish specific signal

• Orthogonal method comparison:

  • Compare protein detection with antibodies targeting different epitopes

  • Correlate protein expression with mRNA levels (RT-PCR)

• Genetic manipulation controls:

  • Overexpression systems to confirm increased signal

  • siRNA/shRNA knockdown to demonstrate reduced signal

Thorough validation is particularly important when studying proteins like FRMD5 where limited research has been published, reducing the reference points for expected results .

How can researchers address non-specific binding when using FRMD5 antibodies in complex tissue samples?

Non-specific binding is a common challenge when working with membrane proteins like FRMD5 in complex tissue samples. To address this issue:

• Optimization of blocking strategy:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 2-3 hours at room temperature

  • Consider adding 0.1-0.3% Triton X-100 to blocking solution to reduce hydrophobic interactions

• Antibody cross-adsorption:

  • Pre-incubate antibody with tissues/cells lacking FRMD5 expression

  • Use species-specific tissue for cross-adsorption to remove cross-reactive antibodies

• Titration approach:

  • Test multiple antibody dilutions (from 1:50 to 1:1000)

  • Optimize secondary antibody concentrations independently

  • Reduce primary antibody incubation time if background persists

• Sample preparation refinements:

  • Fresh tissue fixation with shorter fixation times

  • Extensive washing steps (5-6 washes of 10 minutes each)

  • Use of detergents specific for membrane proteins (e.g., digitonin)

• Signal amplification alternatives:

  • Consider tyramide signal amplification for weak signals

  • Use directly conjugated primary antibodies to eliminate secondary antibody cross-reactivity

These approaches should be systematically tested and documented to establish optimal conditions for each tissue type and experimental system .

What strategies can resolve discrepancies in FRMD5 detection across different experimental techniques?

When facing inconsistent FRMD5 detection between techniques (e.g., positive Western blot but negative immunofluorescence), consider these methodological approaches:

• Epitope accessibility analysis:

  • Different experimental conditions expose different epitopes

  • Try antibodies targeting alternative regions (N-terminal vs. AA 211-312 vs. AA 295-504)

  • Test multiple antibody clones raised against the same region

• Protocol modification for epitope demasking:

  • For formaldehyde-fixed samples, extend antigen retrieval time

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Consider membrane protein-specific extraction buffers (containing mild detergents)

• Protein-protein interaction considerations:

  • FERM domain interactions may shield epitopes in native conditions

  • Denaturing conditions in Western blotting expose epitopes hidden in fixed tissue

• Expression level threshold differences:

  • Western blotting often detects lower expression levels than immunohistochemistry

  • Consider signal amplification systems for techniques with lower sensitivity

• Combined approach validation:

  • Use multiple techniques in parallel (Western blot, IF, IP-MS)

  • Correlate with mRNA expression data from the same samples

  • Include positive control tissues with known high FRMD5 expression

Remember that FRMD5 exists in up to two isoforms, which may be differentially detected by various antibodies and techniques .

How can researchers design experiments to investigate FRMD5's role in cell migration?

To investigate FRMD5's suspected role in cell migration, researchers should consider this comprehensive experimental design:

• Expression manipulation approaches:

  • CRISPR/Cas9 knockout cell lines

  • siRNA/shRNA knockdown (transient and stable)

  • Overexpression of wild-type and domain-specific mutants

  • Rescue experiments with mutant variants

• Quantitative migration assays:

  • Wound healing/scratch assays with time-lapse imaging

  • Transwell migration assays (modifying ECM composition)

  • 3D invasion assays in relevant matrix compositions

  • Single-cell tracking with automated image analysis

• Mechanistic investigations:

  • Actin cytoskeleton visualization (phalloidin staining)

  • Focal adhesion protein co-localization studies

  • Live-cell imaging with fluorescently-tagged FRMD5

  • FRET/BRET assays to identify protein-protein interactions

• Signaling pathway analysis:

  • Phosphorylation status of migration-related signaling molecules

  • Small molecule inhibitors of candidate pathways

  • Proximity ligation assays to detect protein interactions in situ

• Tissue-specific considerations:

  • Primary cell cultures from relevant tissues

  • Organoid migration models

  • In vivo models using conditional knockout approaches

This multi-faceted approach can help establish causal relationships between FRMD5 expression/function and cell migration phenotypes .

What is known about FRMD5's association with neurodevelopmental disorders?

FRMD5 has been associated with neurodevelopmental disorders characterized by eye movement abnormalities and ataxia. Current research suggests several potential mechanisms for this association:

• Cellular mechanisms:

  • FRMD5's membrane localization suggests potential roles in neuronal membrane organization

  • As a cytoskeletal-membrane linker protein, FRMD5 may influence neuronal migration during development

  • Potential involvement in synapse formation and stability

• Genetic evidence:

  • The FRMD5 gene is located on human chromosome 15, a region associated with multiple neurodevelopmental disorders

  • This chromosome houses over 700 genes and comprises nearly 3% of the human genome

  • Genetic disorders linked to this region include Angelman syndrome and Prader-Willi syndrome

• Research approaches to investigate this association:

  • Patient-derived iPSCs differentiated into neural lineages

  • FRMD5 expression analysis in neurodevelopmental disorder brain samples

  • Animal models with FRMD5 mutations assessing neurological phenotypes

  • Structural and functional neuroimaging correlated with FRMD5 variants

• Emerging research directions:

  • Examining FRMD5 expression in cerebellum (ataxia association)

  • Investigating FRMD5's role in oculomotor neuron development

  • Exploring genetic interactions with other neurodevelopmental risk genes

Further research is needed to fully elucidate the mechanistic relationship between FRMD5 dysfunction and neurodevelopmental phenotypes .

How does FRMD5 interact with other cytoskeletal proteins and signaling pathways?

As a FERM domain-containing protein, FRMD5 likely participates in complex interactions with cytoskeletal components and signaling molecules. Current understanding suggests:

• Potential cytoskeletal interaction partners:

  • Actin filaments (direct or indirect binding)

  • Membrane phospholipids (via FERM domain)

  • Transmembrane proteins (receptor tyrosine kinases, adhesion molecules)

  • Other cytoskeletal organizers (ERM family proteins)

• Predicted signaling pathway connections:

  • Rho GTPase signaling (cell migration regulation)

  • Focal adhesion kinase (FAK) pathways

  • Phosphoinositide signaling at the membrane

  • Mechanotransduction pathways

• Experimental approaches to study these interactions:

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity-dependent biotinylation (BioID, APEX)

  • Yeast two-hybrid screening for direct interaction partners

  • Phosphoproteomic analysis following FRMD5 manipulation

• Functional relevance assessment:

  • Subcellular localization studies during different cellular processes

  • Response to cytoskeletal-disrupting agents

  • Activation status of downstream signaling molecules

  • Mechanical property changes in FRMD5-depleted cells

Understanding these interactions will provide insight into FRMD5's cellular functions and potentially reveal therapeutic targets for associated disorders .

What are the emerging techniques for studying FRMD5 expression and localization in tissue-specific contexts?

Advanced techniques for investigating FRMD5 expression and localization in tissue-specific contexts include:

• Single-cell analysis approaches:

  • Single-cell RNA sequencing to identify cell types expressing FRMD5

  • Mass cytometry (CyTOF) with metal-conjugated FRMD5 antibodies

  • Spatial transcriptomics to map FRMD5 expression in tissue architecture

  • CODEX multiplexed imaging for simultaneous detection of multiple markers

• Advanced microscopy techniques:

  • Super-resolution microscopy (STORM, PALM, SIM) for nanoscale localization

  • Expansion microscopy to physically enlarge specimens for enhanced resolution

  • Live-cell lattice light-sheet microscopy for dynamic localization studies

  • Correlative light and electron microscopy for ultrastructural context

• Tissue-specific expression systems:

  • Inducible, cell-type-specific FRMD5 reporters in model organisms

  • Tissue-specific CRISPR knockout models

  • Human tissue organoids for developmental expression studies

  • Patient-derived xenografts for disease-relevant expression patterns

• Emerging antibody-based technologies:

  • Highly multiplexed immunofluorescence (Cyclic IF, CODEX)

  • In situ proximity ligation assays for protein interaction mapping

  • Antibody-based CLARITY tissue clearing compatible techniques

  • Quantum dot-conjugated antibodies for long-term imaging

These emerging methodologies enable researchers to study FRMD5 with unprecedented spatial, temporal, and functional resolution in physiologically relevant contexts .

What criteria should guide the selection of appropriate FRMD5 antibodies for specific research questions?

Selecting the optimal FRMD5 antibody requires careful consideration of multiple technical factors aligned with specific research objectives:

When designing experiments involving FRMD5, researchers should evaluate antibodies based on:

• Validation data in applications matching their planned experiments

• Epitope location relative to functional domains or interaction sites

• Host species compatibility with other antibodies in multiplexed studies

• Batch-to-batch consistency documentation

• Published literature using the specific antibody clone

This strategic approach to antibody selection significantly increases experimental reproducibility and data reliability .

What are promising future directions for FRMD5 research based on current knowledge gaps?

Several promising research directions emerge from current knowledge gaps about FRMD5:

• Functional genomics approaches:

  • Comprehensive CRISPR screens to identify genetic interactions

  • Detailed characterization of FRMD5 promoter regulation

  • Tissue-specific conditional knockout models to assess developmental functions

  • Identification and characterization of additional splice variants

• Structural biology investigations:

  • Cryo-EM studies of FRMD5 in membrane complexes

  • Detailed analysis of FERM domain interactions with binding partners

  • Conformational changes during activation/inactivation cycles

  • Rational design of domain-specific inhibitors

• Disease-relevant research:

  • Comprehensive analysis of FRMD5 variants in neurodevelopmental disorders

  • Investigation of FRMD5 in cell migration during neural development

  • Exploration of potential roles in other disorders involving cytoskeletal regulation

  • Development of FRMD5-focused therapeutic approaches

• Systems biology integration:

  • Multi-omics approaches to place FRMD5 in broader cellular networks

  • Computational modeling of FRMD5's role in cytoskeletal dynamics

  • Temporal analysis of FRMD5 complexes during differentiation processes

  • Machine learning approaches to predict FRMD5 functions from expression patterns

These directions represent significant opportunities to advance understanding of this understudied protein and its potential contributions to human disease .