FAM84A Antibody

What is FAM84A and why is it of interest to researchers?

FAM84A (Family with Sequence Similarity 84, Member A) is a protein that has increasingly gained attention in cancer research. It is primarily studied for its role in cancer development, particularly in papillary thyroid carcinoma (PTC), where it has been identified as an oncogenic factor. Research has shown that FAM84A is significantly upregulated in PTC tissues compared to normal tissues, and this upregulation correlates with larger tumor size, higher lymph node metastasis rates, and advanced TNM staging . The growing evidence of FAM84A's involvement in cancer proliferation and metastasis makes it an important target for both diagnostic and therapeutic research in oncology.

What are the most commonly used applications for FAM84A antibodies?

FAM84A antibodies are versatile research tools employed in several standard laboratory techniques. The most common applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of FAM84A in samples

• Western Blotting (WB): For detecting FAM84A protein expression levels and molecular weight

• Immunohistochemistry (IHC): Particularly on paraffin-embedded sections to visualize FAM84A expression in tissue samples

Additional applications may include immunofluorescence (IF) on both cultured cells and paraffin sections, and immunocytochemistry (ICC). The selection of the appropriate application depends on the specific research question, sample type, and desired outcome of the experiment.

How should researchers select the appropriate FAM84A antibody for their experiments?

When selecting a FAM84A antibody, researchers should consider several key factors:

• Binding specificity: Determine which region of FAM84A the antibody recognizes. Some antibodies target specific amino acid sequences (e.g., AA 1-292, AA 151-250, or AA 15-144), while others target the N-terminal region .

• Species reactivity: Verify that the antibody reacts with your species of interest. Available antibodies show reactivity against human, mouse, rat, and sometimes additional species including cow, dog, guinea pig, rabbit, zebrafish, bat, chicken, monkey, and pig .

• Host species and clonality: Consider whether a monoclonal (more specific) or polyclonal (broader epitope recognition) antibody is more suitable for your application. Mouse monoclonal and rabbit polyclonal FAM84A antibodies are commonly available .

• Conjugation: Determine if you need a conjugated or unconjugated antibody based on your detection method.

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, ELISA, etc.).

What are the recommended storage and handling conditions for FAM84A antibodies?

Although specific storage conditions may vary between manufacturers, general best practices for FAM84A antibodies include:

• Temperature: Store antibodies at -20°C for long-term storage or at 4°C for short-term storage (typically less than one month)

• Aliquoting: Upon first use, aliquot the antibody into smaller volumes to avoid repeated freeze-thaw cycles

• Freeze-thaw cycles: Minimize freeze-thaw cycles as they can lead to antibody degradation and loss of activity

• Working dilutions: Prepare working dilutions just before use and discard any unused diluted antibody

• Sterility: Use sterile techniques when handling antibodies to prevent contamination

• Buffer considerations: Some antibodies may contain preservatives like sodium azide, which can inhibit certain enzymatic reactions

Always refer to the manufacturer's specific recommendations for optimal results.

How can FAM84A antibodies be utilized to study its role in the Wnt/β-catenin signaling pathway?

FAM84A has been identified as a significant component in the Wnt/β-catenin signaling pathway, particularly in cancer progression. To investigate this relationship, researchers can employ several approaches:

• Co-immunoprecipitation assays: Use FAM84A antibodies to pull down protein complexes and probe for β-catenin and other Wnt pathway components to establish physical interactions

• Western blot analysis: Employ FAM84A antibodies alongside antibodies for key Wnt pathway proteins (β-catenin, N-cadherin, vimentin, E-cadherin) to track changes in their expression levels following FAM84A knockdown or overexpression

• Immunofluorescence analysis: Use FAM84A antibodies in conjunction with β-catenin antibodies to visualize subcellular localization, particularly nuclear translocation of β-catenin which is indicative of Wnt pathway activation

• TOP/FOP luciferase reporter assays: Measure Wnt/β-catenin transcriptional activity following manipulation of FAM84A expression to establish functional relevance

Research has shown that knockdown of FAM84A inhibits the expression of nucleus β-catenin and reduces TOP/FOP transcriptional activity, suggesting that FAM84A positively regulates the Wnt/β-catenin pathway .

What experimental approaches can be used to investigate the relationship between FAM84A and miR-874-3p?

The relationship between FAM84A and miR-874-3p represents an important regulatory mechanism in cancer biology. To study this interaction, researchers can implement several methodologies:

• Dual-luciferase reporter assay: The most direct method to confirm miRNA targeting, where the wild-type and mutated FAM84A 3'UTR are cloned into reporter vectors and co-transfected with miR-874-3p mimics to measure changes in luciferase activity

• qRT-PCR and Western blot analysis: Transfect cells with miR-874-3p mimics and measure changes in FAM84A mRNA and protein expression levels

• RNA immunoprecipitation (RIP) assay: Use antibodies against components of the RNA-induced silencing complex (like Ago2) to immunoprecipitate miRNA-target complexes and detect FAM84A mRNA enrichment

• RNA pull-down assay: Employ biotinylated miR-874-3p to capture FAM84A mRNA and demonstrate direct interaction

• Expression correlation analysis: Analyze public databases like TCGA to identify inverse correlations between miR-874-3p and FAM84A expression levels in patient samples

Studies have confirmed that FAM84A is a direct downstream target of miR-874-3p, with overexpression of miR-874-3p significantly reducing FAM84A expression levels .

What strategies can be employed to verify FAM84A antibody specificity for research applications?

Ensuring antibody specificity is crucial for generating reliable research data. For FAM84A antibodies, consider these validation approaches:

• Knockdown/knockout controls: Perform Western blot or immunostaining on samples where FAM84A has been knocked down (siRNA, shRNA) or knocked out (CRISPR-Cas9) to confirm signal reduction or elimination

• Overexpression controls: Compare signal in cells overexpressing recombinant FAM84A versus control cells

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

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of FAM84A to confirm consistent results

• Cross-reactivity testing: Test the antibody against closely related family members to ensure specificity

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein

These approaches collectively provide strong evidence for antibody specificity and reliability in experimental settings.

How can FAM84A antibodies be optimized for detecting epithelial-mesenchymal transition (EMT) in cancer research?

FAM84A has been implicated in promoting epithelial-mesenchymal transition (EMT), a critical process in cancer metastasis. To optimize detection of EMT changes related to FAM84A, researchers should:

• Multiplex immunofluorescence: Combine FAM84A antibodies with antibodies against established EMT markers (E-cadherin, N-cadherin, vimentin) to simultaneously visualize expression changes in single cells

• Sequential immunoblotting: Perform Western blot analysis on the same membrane for FAM84A and EMT markers to establish correlation in expression levels

• Morphological analysis: Combine FAM84A immunostaining with morphological analysis to correlate FAM84A expression with cell shape changes characteristic of EMT

• Functional EMT assays: Use FAM84A antibodies in conjunction with migration and invasion assays to correlate protein expression with functional outcomes

• Time-course experiments: Track FAM84A and EMT marker expression during induced EMT to establish temporal relationships

Research has demonstrated that knockdown of FAM84A in cancer cell lines decreases the expression of mesenchymal markers (vimentin, N-cadherin) while increasing epithelial marker E-cadherin expression, confirming its role in promoting EMT .

What considerations should be made when designing FAM84A knockdown or overexpression experiments?

When manipulating FAM84A expression levels for functional studies, researchers should address several key considerations:

• Selection of knockdown/overexpression method:

  • For transient knockdown: siRNA transfection is effective for short-term studies (3-5 days)

  • For stable knockdown: shRNA constructs delivered via lentiviral vectors allow for long-term studies and in vivo experiments

  • For overexpression: full-length FAM84A cDNA expression vectors with appropriate tags for detection

• Validation of expression changes:

  • Always confirm knockdown or overexpression efficiency at both mRNA level (qRT-PCR) and protein level (Western blot)

  • Aim for at least 70-80% knockdown efficiency for meaningful functional studies

• Appropriate controls:

  • Include non-targeting siRNA/shRNA controls that activate RISC complex but do not target any known genes

  • For overexpression, use empty vector controls with the same promoter and selection markers

• Rescue experiments:

  • Consider performing rescue experiments by re-expressing siRNA-resistant FAM84A in knockdown cells to confirm specificity of observed phenotypes

• Cell line selection:

  • Choose cell lines with detectable baseline FAM84A expression for knockdown studies

  • Select cell lines with low endogenous expression for overexpression studies

  • Include multiple cell lines to establish generalizability of findings

What are the optimal protocols for using FAM84A antibodies in Western blotting?

For optimal Western blot results with FAM84A antibodies, researchers should follow these recommendations:

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • For nuclear and cytoplasmic fractionation studies, use specialized extraction kits to maintain compartment integrity

• Protein loading:

  • Load 20-40 μg of total protein per lane

  • Include appropriate loading controls (GAPDH for total lysate, Histone H3 for nuclear fraction)

• SDS-PAGE conditions:

  • Use 10-12% polyacrylamide gels for optimal separation

  • Run at 80-120V to ensure proper protein migration

• Transfer parameters:

  • Transfer to PVDF membrane (preferred over nitrocellulose for FAM84A detection)

  • Use wet transfer at 100V for 60-90 minutes or overnight at 30V at 4°C

• Blocking conditions:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody: Dilute FAM84A antibody 1:1000-1:1500 in blocking buffer and incubate overnight at 4°C

  • Secondary antibody: Incubate for 2 hours at room temperature

• Detection method:

  • Use enhanced chemiluminescence (ECL) detection system for visualization

  • For quantitative analysis, consider fluorescent secondary antibodies and imaging

How can researchers effectively interpret conflicting FAM84A expression data across different cancer types?

When faced with conflicting FAM84A expression data across cancer types or studies, researchers should implement a systematic approach to data interpretation:

• Data normalization assessment:

  • Evaluate the normalization methods used across studies (housekeeping genes, global normalization)

  • Determine if different reference genes might contribute to apparent discrepancies

• Technical platform considerations:

  • Compare detection methods (antibody-based vs. RNA-based techniques)

  • Consider sensitivity and specificity differences between platforms (microarray vs. RNA-seq vs. qPCR vs. Western blot)

• Sample composition analysis:

  • Assess tumor purity and stromal content differences

  • Consider cell type heterogeneity within samples

• Context-dependent regulation:

  • Analyze FAM84A in relation to tissue-specific regulatory networks

  • Consider that FAM84A may have different roles in different cellular contexts

• Validation approach:

  • Design experiments that directly compare FAM84A expression across multiple cancer types using standardized methods

  • Use tissue microarrays with multiple cancer types processed simultaneously

• Meta-analysis strategy:

  • Perform or consult meta-analyses that integrate data across multiple studies with appropriate statistical adjustments

  • Use public databases like TCGA and GEO to investigate larger sample sizes

How can chromatin immunoprecipitation (ChIP) be combined with FAM84A research to understand its transcriptional regulation?

Understanding the transcriptional regulation of FAM84A can provide insights into its dysregulation in disease states. Chromatin immunoprecipitation (ChIP) offers a powerful approach:

• Identification of transcription factors:

  • Perform ChIP using antibodies against predicted transcription factors that might regulate FAM84A

  • Follow with qPCR targeting the FAM84A promoter region

  • Alternatively, perform ChIP-seq to identify genome-wide binding patterns

• Histone modification analysis:

  • Use ChIP with antibodies against histone modifications (H3K4me3, H3K27ac, H3K27me3) to assess the chromatin state at the FAM84A locus

  • Compare histone modification patterns between normal and cancer tissues

• Integration with expression data:

  • Correlate ChIP data with FAM84A expression levels to establish functional relevance

  • Use computational approaches to identify transcription factor binding motifs in regulatory regions

• Experimental validation:

  • Perform luciferase reporter assays with wild-type and mutated FAM84A promoter constructs

  • Use CRISPR-based techniques to modify putative regulatory elements

This integrated approach can reveal the upstream mechanisms controlling FAM84A expression and potentially identify new therapeutic targets for modulating its expression.

What emerging techniques might enhance the study of FAM84A protein interactions in signaling networks?

Several cutting-edge technologies can provide deeper insights into FAM84A's role in signaling networks:

• Proximity labeling proteomics:

  • BioID or APEX2 fusion constructs with FAM84A to identify proximal proteins in living cells

  • TurboID for faster labeling kinetics to capture transient interactions

• Protein-protein interaction mapping:

  • Co-immunoprecipitation followed by mass spectrometry (Co-IP-MS)

  • Crosslinking mass spectrometry (XL-MS) to capture direct interaction interfaces

• Live-cell imaging techniques:

  • FRET (Förster Resonance Energy Transfer) to visualize FAM84A interactions in real-time

  • Split fluorescent protein complementation assays to confirm specific interactions

• High-throughput screening approaches:

  • CRISPR screens to identify synthetic lethal interactions with FAM84A

  • Drug screening in FAM84A-high versus FAM84A-low expressing cells

• Spatial proteomics:

  • Multiplexed immunofluorescence to map FAM84A localization relative to other signaling components

  • Imaging mass cytometry for higher-dimensional protein interaction mapping

These techniques collectively can provide a systems-level understanding of FAM84A function within complex signaling networks.

How can single-cell analysis techniques be applied to study heterogeneity in FAM84A expression?

Single-cell approaches offer unprecedented resolution for understanding FAM84A expression heterogeneity in complex tissues:

• Single-cell RNA sequencing (scRNA-seq):

  • Profile FAM84A expression across individual cells in tumors to identify subpopulations

  • Correlate with cell states and other markers to establish functional relevance

  • Perform trajectory analysis to map FAM84A expression changes during cellular differentiation or EMT

• Single-cell proteomics:

  • Use mass cytometry (CyTOF) with FAM84A antibodies to quantify protein at single-cell resolution

  • Employ single-cell Western blotting for protein isoform analysis

• Spatial transcriptomics:

  • Combine in situ hybridization techniques with single-cell resolution to map FAM84A expression within tissue architecture

  • Correlate spatial expression with histopathological features

• Functional heterogeneity assessment:

  • Combine FAM84A detection with functional assays at single-cell level

  • Use microfluidic systems to isolate and characterize FAM84A-expressing cells

These approaches can reveal how FAM84A expression varies across cell types and states, potentially identifying specific cellular contexts where targeting FAM84A might be most effective.

How can FAM84A antibodies be optimized for potential diagnostic applications in cancer?

Given FAM84A's upregulation in certain cancers, optimizing antibodies for diagnostic applications requires several considerations:

• Antibody validation for diagnostics:

  • Extensive testing across diverse patient samples to establish sensitivity and specificity

  • Comparison with gold standard diagnostic methods

  • Standardization of staining protocols across laboratories

• Scoring system development:

  • Create quantitative scoring systems for FAM84A immunohistochemistry

  • Establish clinically relevant cutoff values for "high" versus "low" expression

  • Correlate expression levels with clinical outcomes

• Multiplex approaches:

  • Combine FAM84A detection with other diagnostic markers for improved accuracy

  • Develop multiplexed immunofluorescence panels including FAM84A and established cancer markers

• Automation considerations:

  • Adapt protocols for automated immunostaining platforms

  • Develop digital pathology algorithms for quantitative FAM84A assessment

The potential diagnostic value of FAM84A has been demonstrated in PTC, where higher expression correlates with larger tumor size, lymph node metastasis, and advanced TNM stage .

What considerations should be made when designing FAM84A-targeted therapeutic approaches?

As research reveals FAM84A's role in promoting cancer progression, several approaches for therapeutic targeting can be considered:

• Direct protein targeting:

  • Develop small molecule inhibitors that disrupt FAM84A protein interactions

  • Design peptides that interfere with critical FAM84A binding domains

• Expression modulation:

  • Deliver miR-874-3p mimics to downregulate FAM84A expression

  • Develop antisense oligonucleotides or siRNA therapeutics targeting FAM84A mRNA

• Pathway intervention:

  • Target downstream effectors in the Wnt/β-catenin pathway activated by FAM84A

  • Develop combination approaches targeting both FAM84A and its signaling partners

• Biomarker-guided therapy:

  • Use FAM84A expression as a predictive biomarker for response to targeted therapies

  • Develop companion diagnostics with FAM84A antibodies for patient stratification

• Delivery considerations:

  • Design tumor-specific delivery systems for FAM84A-targeting therapeutics

  • Consider tissue-specific expression patterns to minimize off-target effects

These approaches represent promising avenues for translating basic FAM84A research into clinical applications, particularly for cancers where FAM84A overexpression drives disease progression.