FAM83G Antibody, Biotin conjugated

What is FAM83G and what biological pathways does it regulate?

FAM83G (Family with sequence similarity 83, member G), also known as PAWS1 (Protein Associated with SMAD1), is a substrate for type I BMP receptor kinase involved in the regulation of target genes within the BMP signaling pathway. It also regulates expression of several non-BMP target genes, suggesting its involvement in multiple signaling cascades . FAM83G belongs to the FAM83 family of proteins that anchor isoforms of the CK1 family of serine/threonine kinases to specific subcellular compartments through the conserved DUF1669 domain .

Functionally, FAM83G forms a complex with CK1α that is critical for the activation of canonical WNT signalling . This interaction appears essential for normal development, as disruptions in FAM83G-CK1α binding are associated with pathological conditions including palmoplantar keratoderma and developmental abnormalities. Recent studies have also revealed FAM83G's involvement in odontogenesis (tooth development), further expanding our understanding of its biological significance .

What applications is biotin-conjugated FAM83G antibody suitable for?

Biotin-conjugated FAM83G antibody has been validated for several research applications, with ELISA being the primary validated application . The biotin conjugation provides significant advantages for detection sensitivity due to the strong interaction between biotin and streptavidin, which allows for signal amplification in detection systems.

While primarily validated for ELISA, the antibody may also be suitable for applications where biotin-streptavidin interactions provide advantages, including:

• Immunohistochemistry with streptavidin-based detection systems

• Flow cytometry with streptavidin-conjugated fluorophores

• Immunoprecipitation using streptavidin-coated beads

• Western blotting with streptavidin-HRP detection systems

The unconjugated version of the antibody has been validated for additional applications including immunohistochemistry on paraffin-embedded tissues (IHC-P), western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF), suggesting similar applications may be possible with the biotin-conjugated version after appropriate validation .

What is the proper storage and handling protocol for maintaining antibody activity?

To maintain optimal activity of the biotin-conjugated FAM83G antibody, proper storage and handling are essential. The antibody should be stored at -20°C or -80°C upon receipt . It's critical to avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce its effectiveness. The antibody is provided in liquid form, typically in a buffer containing preservatives and stabilizers including:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 as a preservative

For long-term storage, aliquoting the antibody into smaller volumes before freezing is recommended to avoid repeated freeze-thaw cycles. When handling the antibody, maintain sterile conditions to prevent contamination. When diluting for experimental use, use fresh, sterile buffers, and optimize the dilution ratio based on your specific application and detection system.

How can FAM83G antibodies be utilized to investigate WNT signaling pathway disruptions?

FAM83G forms a critical complex with CK1α that is essential for the activation of canonical WNT signaling . Using biotin-conjugated FAM83G antibodies, researchers can elucidate disruptions in this pathway through several methodological approaches:

• Co-immunoprecipitation studies: Researchers can use the antibody to pull down FAM83G and associated proteins, then assess the presence of CK1α to determine if their interaction is intact. This is particularly relevant when studying mutations like R265P that disrupt this interaction .

• Immunofluorescence co-localization: Pairing the biotin-conjugated FAM83G antibody with antibodies against WNT pathway components allows visualization of their spatial relationships within cells under normal and pathological conditions.

• Chromatin immunoprecipitation (ChIP) assays: For investigating how FAM83G affects gene expression in the WNT pathway, biotin-conjugated antibodies can help identify genomic regions where FAM83G-containing complexes interact with DNA.

• Proximity ligation assays: This technique can quantify protein-protein interactions between FAM83G and CK1α under various conditions, enabling the study of how treatments or mutations affect this critical interaction.

For quantitative assessment of WNT pathway activation, researchers should consider downstream readouts like β-catenin nuclear translocation or expression of WNT target genes in conjunction with FAM83G antibody staining or immunoprecipitation.

What considerations should be made when using FAM83G antibodies in studies of palmoplantar keratoderma?

Palmoplantar keratoderma (PPK) is associated with mutations in FAM83G, particularly variants that disrupt its interaction with CK1α and subsequent WNT signaling . When using FAM83G antibodies in PPK research, several methodological considerations are important:

• Epitope accessibility: Mutations in FAM83G (like R265P) may alter protein conformation, potentially affecting antibody binding. Researchers should verify that their antibody's epitope is not within or affected by the mutated region. The biotin-conjugated FAM83G antibody available is raised against recombinant protein FAM83G (464-615AA), which may not be affected by mutations in other regions like R265 .

• Patient-derived samples: When working with patient samples, controls should include both healthy tissue and, if possible, tissue from unaffected areas of the same patient to account for background genetic variation.

• Protein stability analysis: As demonstrated with the R265P variant, mutations can affect protein stability . Cycloheximide chase experiments using the antibody can track degradation rates of mutant versus wild-type FAM83G.

• Fibroblast culture considerations: When analyzing patient-derived fibroblasts, standardized culture conditions are essential as described in the literature (DMEM supplemented with 20% FBS, L-glutamine, penicillin, and streptomycin) .

• Complementary analysis: Combining FAM83G antibody staining with assessments of downstream WNT signaling components provides more comprehensive insights into pathogenic mechanisms.

When designing experiments, consider that FAM83G mutations in PPK patients may affect not only protein function but also expression levels, localization, and stability, all of which can be assessed using the antibody in different experimental contexts.

How does FAM83G interact with the odontogenic differentiation pathway?

Recent research has revealed an intriguing relationship between FAM83G and odontogenesis (tooth development), particularly in relation to Piezo1-mediated mechanotransduction . When investigating this pathway, researchers should consider the following methodological approaches:

• Expression analysis during differentiation: Using FAM83G antibodies to track protein expression changes during dental papilla cell (DPC) differentiation can provide insights into temporal regulation. RNA-seq data indicates that FAM83G is differentially expressed during odontogenic differentiation and appears to be negatively regulated by Piezo1 .

• Knockdown studies: siRNA-mediated knockdown of FAM83G has been shown to upregulate odontogenic markers including DSPP, DMP1, and ALP. Antibodies can be used to confirm knockdown efficiency at the protein level and to monitor changes in downstream targets .

• Mechanotransduction pathway analysis: The Piezo1-FAM83G axis represents a novel mechanism in odontogenesis. When Piezo1 is activated by compounds like Yoda1, FAM83G protein levels decrease, suggesting a regulatory relationship that can be monitored using the antibody .

• Mineralization assays: Following FAM83G knockdown, increased mineralization nodule formation occurs, which can be assessed alongside FAM83G protein levels using antibody-based techniques .

The following table summarizes key findings from knockdown studies that researchers should consider when designing FAM83G-focused odontogenesis experiments:

This suggests FAM83G acts as a negative regulator of odontogenic differentiation, making it a potential therapeutic target for dental regenerative approaches .

What optimization steps are necessary for using FAM83G biotin-conjugated antibody in ELISA?

Optimizing ELISA protocols with biotin-conjugated FAM83G antibody requires careful attention to several parameters:

• Antibody dilution optimization: Though the manufacturer may provide a recommended range, it's essential to perform a titration series (typically 1:100, 1:500, 1:1000, 1:5000) to determine the optimal dilution that provides maximum specific signal with minimal background. The biotin-conjugated FAM83G antibody has been validated for ELISA applications, but optimal dilutions may vary by experimental context .

• Detection system selection: Since the antibody is biotin-conjugated, the detection system should utilize streptavidin linked to an appropriate reporter (e.g., streptavidin-HRP for colorimetric detection). The concentration of streptavidin-reporter conjugate should also be optimized.

• Blocking optimization: To minimize nonspecific binding, test different blocking agents (BSA, non-fat milk, commercial blocking buffers) at various concentrations (2-5%) and incubation times (1-2 hours).

• Sample preparation consideration: When detecting FAM83G in complex biological samples, pretreatment steps may be necessary. For cell lysates, ensure complete lysis using buffers compatible with the ELISA format. For tissue samples, homogenization protocols should be standardized.

• Validation controls: Include:

  • Positive control: Recombinant FAM83G protein or lysates from cells known to express FAM83G

  • Negative control: Lysates from FAM83G knockout cells or tissues

  • Antibody controls: Include wells without primary antibody and without sample to assess non-specific binding

• Standard curve development: If performing quantitative ELISA, prepare a standard curve using recombinant FAM83G protein spanning the expected concentration range of your samples.

Allow sufficient incubation time (typically 1-2 hours at room temperature or overnight at 4°C) for the antibody binding step to ensure optimal signal development.

What are the key considerations for using FAM83G antibodies in co-immunoprecipitation studies investigating protein interactions?

Co-immunoprecipitation (Co-IP) studies are particularly valuable for investigating FAM83G's interactions with partners like CK1α. When using FAM83G antibodies for Co-IP, consider these methodological principles:

• Antibody orientation: Since the antibody is biotin-conjugated, it can be immobilized on streptavidin-coated beads. Alternatively, if using protein A/G beads, a bridging antibody (anti-biotin) may be necessary.

• Cell lysis conditions: FAM83G interactions, particularly with CK1α, are critical for its function . Use lysis buffers that preserve protein-protein interactions:

  • Start with mild non-ionic detergents (0.5-1% NP-40 or Triton X-100)

  • Include protease and phosphatase inhibitors

  • Maintain physiological salt concentrations (~150 mM NaCl)

  • Consider preserving post-translational modifications that may be important for interactions

• Controls for validation:

  • Input control: Sample of lysate before immunoprecipitation

  • Negative control: Non-specific antibody of the same isotype

  • Blocking peptide control: Pre-incubation of antibody with immunizing peptide

  • Reverse Co-IP: Use antibody against suspected interacting partner to pull down FAM83G

• Detection strategy: When probing for co-precipitated proteins, avoid detection interference from the heavy and light chains of the immunoprecipitating antibody. Options include:

  • Using HRP-conjugated protein A/G for detection

  • Using light chain-specific secondary antibodies

  • Running non-reducing SDS-PAGE to keep antibody chains intact

• Confirming specificity: Validate interactions with reciprocal Co-IPs and through additional methods such as proximity ligation assays or FRET.

For known FAM83G variants like R265P that disrupt CK1α binding , Co-IP can quantitatively assess the degree of interaction loss, providing mechanistic insights into pathogenic processes.

How can FAM83G antibodies be effectively utilized in immunofluorescence studies?

While the biotin-conjugated FAM83G antibody is primarily validated for ELISA , similar antibodies have been successfully used in immunofluorescence applications . For adapting the biotin-conjugated antibody to immunofluorescence, consider these methodological approaches:

• Sample preparation:

  • Fixation: For cellular localization studies, 4% paraformaldehyde for 15-20 minutes is typically suitable

  • Permeabilization: 0.1-0.5% Triton X-100 for intracellular antigens

  • Blocking: 5-10% normal serum from the same species as the secondary antibody

• Antibody application strategy:

  • Primary detection: Use a 1:50 to 1:200 dilution range as a starting point for optimization

  • Detection system: Utilize streptavidin conjugated to a fluorophore (e.g., Alexa Fluor 488, 594) to bind the biotin-conjugated primary antibody

  • Counter-staining: DAPI for nuclear visualization; consider co-staining with markers for subcellular compartments or interacting partners

• Controls for validation:

  • Negative control: Omit primary antibody

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • Cellular controls: Use cells with known FAM83G expression (positive) and FAM83G knockdown or knockout cells (negative)

• Imaging parameters:

  • Capture multiple fields to ensure representative sampling

  • Include z-stack imaging for accurate subcellular localization

  • Use consistent exposure settings when comparing experimental conditions

For co-localization studies with potential interacting partners like CK1α, select fluorophores with minimal spectral overlap and include appropriate controls for each channel. The subcellular localization of FAM83G should be primarily cytoplasmic based on previous studies .

When studying mutants like R265P, immunofluorescence can reveal changes in protein localization or expression levels that contribute to pathological mechanisms .

How can researchers address specificity concerns when using FAM83G antibodies?

Ensuring antibody specificity is crucial for generating reliable data. For FAM83G antibodies, consider these methodological approaches to address specificity concerns:

• Validation in knockout/knockdown systems:

  • Perform western blotting, immunofluorescence, or ELISA using samples from FAM83G knockout cells (like FAM83G^-/- DLD1 cells mentioned in the literature ) compared with wild-type cells

  • Use siRNA-mediated knockdown of FAM83G (as demonstrated in the literature ) as a control to verify the reduction in signal correlates with reduced FAM83G expression

• Epitope mapping and cross-reactivity assessment:

  • Review the immunogen information: The biotin-conjugated antibody is raised against recombinant FAM83G protein (464-615AA)

  • Assess potential cross-reactivity with other FAM83 family members through sequence alignment of the immunizing peptide region

  • Consider testing the antibody against recombinant proteins of related family members

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide before application

  • A specific antibody will show diminished or absent signal compared to the non-competed condition

• Multiple antibody approach:

  • Compare results using antibodies raised against different epitopes of FAM83G

  • Concordant results across antibodies increase confidence in specificity

• Mass spectrometry validation:

  • For immunoprecipitation applications, confirm the identity of pulled-down proteins through mass spectrometry

The polyclonal nature of the biotin-conjugated FAM83G antibody provides recognition of multiple epitopes, potentially increasing sensitivity but also raising the possibility of increased background. Careful optimization of dilution and thorough validation are particularly important for polyclonal antibodies.

What strategies can optimize detection sensitivity when working with low FAM83G expression levels?

When FAM83G expression is low or difficult to detect, several methodological strategies can enhance sensitivity:

• Signal amplification systems:

  • Leverage the biotin-streptavidin system's natural amplification capacity by using streptavidin conjugated to polymeric detection systems

  • Consider tyramide signal amplification (TSA) compatible with biotin-streptavidin systems for immunohistochemistry and immunofluorescence

  • For ELISA, use high-sensitivity substrates like QuantaBlu or SuperSignal ELISA

• Sample enrichment techniques:

  • Immunoprecipitation to concentrate FAM83G before detection

  • For tissue samples, use laser capture microdissection to isolate cell populations with higher expression

  • Consider cell fractionation to isolate subcellular compartments where FAM83G is concentrated

• Optimized protein extraction:

  • Use extraction buffers containing chaotropic agents for difficult-to-extract proteins

  • For membrane-associated fractions, include appropriate detergents

  • Add protease inhibitors to prevent degradation during extraction

• Detection system optimization:

  • For western blotting, use PVDF membranes (higher protein binding capacity than nitrocellulose)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize blocking conditions to minimize background while preserving specific signal

• Protein stability considerations:

  • Some FAM83G variants (like R265P) have reduced stability

  • Consider treating cells with proteasome inhibitors before extraction to prevent degradation

  • Process samples quickly and maintain cold conditions throughout

For quantitative applications, digital detection platforms like digital ELISA (e.g., Simoa) or digital droplet PCR for mRNA detection can provide significantly lower detection limits than traditional methods.

What approaches are recommended for optimizing FAM83G antibody dilutions for different experimental applications?

Proper antibody dilution is critical for balancing specificity, sensitivity, and resource efficiency. For biotin-conjugated FAM83G antibody, systematic optimization approaches include:

• Systematic titration strategy:

  • For ELISA: Test a logarithmic dilution series (1:100, 1:500, 1:1000, 1:5000, 1:10000)

  • For immunofluorescence: Start with more concentrated dilutions (1:50, 1:100, 1:200, 1:500)

  • For western blotting: Test a range from 1:200 to 1:2000

• Signal-to-background ratio analysis:

  • For each dilution, calculate the ratio between specific signal and background

  • Plot the signal-to-background ratio against antibody dilution to identify the optimal point

  • The optimal dilution provides maximum signal with minimal background

• Application-specific considerations:

  • ELISA: Coating concentration of capture antibody or antigen affects optimal detection antibody dilution

  • Immunofluorescence: Fixation method influences epitope accessibility and optimal dilution

  • Western blotting: Transfer efficiency and blocking conditions impact optimal concentration

• Cross-application standardization:

  • When transitioning between applications, start with a dilution 2-5× more concentrated than the established optimal dilution for the previous application

  • For biotin-conjugated antibodies, the detection system (streptavidin conjugate) concentration must also be optimized

• Sample-dependent optimization:

  • Different tissue or cell types may require different antibody dilutions due to varying expression levels and matrix effects

  • Create a standardized positive control sample to use across experiments for consistent optimization

When documenting optimization, record not just the dilution ratio but also the starting concentration of the antibody (when known) and the final concentration used in the assay, as antibody batches may vary in concentration.

How are FAM83G antibodies contributing to understanding palmoplantar keratoderma pathogenesis?

FAM83G antibodies have become instrumental in elucidating the molecular mechanisms underlying palmoplantar keratoderma (PPK) pathogenesis through several research approaches:

• Mutation-specific functional analysis:

  • The FAM83G R265P variant identified in a 60-year-old female PPK patient shows poor stability and loss of interaction with CK1α

  • Antibody-based studies demonstrated that this mutation attenuates WNT signalling, linking the molecular defect to the clinical phenotype

  • Future research could use FAM83G antibodies to screen for other mutations that disrupt similar functional domains

• Patient-derived cell studies:

  • FAM83G antibodies have enabled characterization of protein expression and function in skin fibroblasts derived from PPK patients

  • This approach bridges the gap between genetic findings and cellular phenotypes

  • Comparative analyses of FAM83G protein levels, stability, and interactions in patient versus control cells provide mechanistic insights

• Signaling pathway interconnections:

  • Antibody-based investigations have revealed that FAM83G links BMP and WNT pathways, suggesting convergent signaling mechanisms in skin development and maintenance

  • These findings point to potential therapeutic targets beyond FAM83G itself

• Structure-function relationships:

  • By using antibodies to study expression and stability of various FAM83G mutants, researchers have mapped critical functional domains

  • The DUF1669 domain in particular has been identified as essential for CK1α binding and subsequent WNT signaling activation

Future research directions may include:

• Using FAM83G antibodies to identify additional interacting partners in keratinocytes

• Investigating potential compensatory mechanisms in FAM83G-deficient cells

• Screening therapeutic compounds that stabilize mutant FAM83G protein or restore its interactions

What are the emerging applications of FAM83G antibodies in dental and craniofacial research?

Recent studies have revealed an unexpected role for FAM83G in odontogenesis, opening new research avenues where FAM83G antibodies serve as valuable tools:

• Mechanotransduction pathway investigation:

  • FAM83G has been identified as a downstream target of Piezo1-mediated mechanotransduction in dental papilla cells (DPCs)

  • Antibody-based studies revealed that Yoda1 (a Piezo1 activator) inhibits FAM83G expression at both mRNA and protein levels

  • This regulation appears to be functionally significant, as FAM83G knockdown promotes odontogenic differentiation

• Developmental expression profiling:

  • FAM83G antibodies enable precise temporal and spatial mapping of protein expression during tooth development

  • Such expression profiles can reveal critical developmental windows where FAM83G function is most significant

  • Comparative analysis with other developmental markers may uncover new regulatory relationships

• Tissue engineering applications:

  • The finding that FAM83G negatively regulates odontogenic differentiation suggests modulating its expression could enhance dental tissue regeneration

  • Antibodies are essential for monitoring the effectiveness of such interventions

• Cross-tissue comparative analysis:

  • FAM83G's dual role in skin (PPK pathogenesis) and dental development suggests common developmental principles

  • Antibody-based comparative studies across ectodermal tissues may reveal shared regulatory mechanisms

This research area is particularly promising given the quantitative data showing:

• Knockdown of FAM83G significantly upregulates odontogenic markers (DSPP, DMP1, ALP)

• Alizarin red staining revealed enhanced mineralization nodule formation following FAM83G knockdown

• ALP activity increased significantly after FAM83G depletion

Future work may focus on:

• Determining whether FAM83G mutations in PPK patients correlate with dental abnormalities

• Investigating FAM83G's role in other craniofacial developmental processes

• Exploring therapeutic applications targeting FAM83G in dental regenerative medicine

How might advances in antibody technology enhance future FAM83G research?

Emerging antibody technologies are poised to transform FAM83G research in several key areas:

• Single-cell antibody-based proteomics:

  • Mass cytometry (CyTOF) using metal-conjugated FAM83G antibodies could enable high-dimensional analysis of protein expression at single-cell resolution

  • This approach would reveal cell-to-cell heterogeneity in FAM83G expression and signaling within complex tissues

  • Integration with single-cell transcriptomics would provide unprecedented insights into FAM83G regulation

• Intrabody development:

  • Engineered antibody fragments (intrabodies) targeting FAM83G could be expressed within living cells

  • This would enable real-time visualization of FAM83G localization and dynamics

  • Functionalized intrabodies could selectively disrupt specific FAM83G interactions (e.g., with CK1α) to dissect its functional domains

• Nanobody technology:

  • Single-domain antibodies (nanobodies) against FAM83G offer advantages in size (allowing access to restricted epitopes) and stability

  • Their small size enables super-resolution microscopy applications for visualizing FAM83G-containing complexes below the diffraction limit

  • Nanobodies can be genetically encoded for live-cell imaging of FAM83G

• Antibody-based proximity labeling:

  • FAM83G antibodies conjugated to promiscuous biotin ligases (BioID) or peroxidases (APEX) could identify proteins in close proximity to FAM83G

  • This approach would map the complete "interactome" of FAM83G in different cellular contexts

  • Comparing interactomes between wild-type and mutant FAM83G would reveal how pathogenic variants alter protein interaction networks

• Conformation-specific antibodies:

  • Development of antibodies that specifically recognize active vs. inactive conformations of FAM83G

  • These would enable quantification of functionally relevant protein states rather than just total protein levels

  • Particularly valuable for studying how mutations affect protein structure and function

These technological advances will likely contribute to more nuanced understanding of how FAM83G functions in different biological contexts, potentially revealing new therapeutic approaches for associated disorders like palmoplantar keratoderma and identifying novel applications in tissue engineering.