FAM83D Antibody, Biotin conjugated

What is FAM83D and what functional roles make it significant for cancer research?

FAM83D (Family with Sequence Similarity 83, Member D) is a protein that plays critical roles in cell division, proliferation, and migration. It localizes to the mitotic spindle and is essential for proper spindle positioning and timely cell division through interaction with protein kinase CK1α . FAM83D has gained significant research interest due to its overexpression in various cancers including hepatocellular carcinoma (HCC), breast cancer, metastatic lung adenocarcinomas, and ovarian cancer .

At the molecular level, FAM83D functions as an oncogenic protein by promoting cell proliferation and motility while inhibiting apoptosis. It interacts with the tumor suppressor F-box protein FBXW7, potentially downregulating its activity and contributing to tumorigenesis . High expression of FAM83D correlates with advanced clinical stage, higher tumor grade, and poor prognosis in HCC patients . Additionally, FAM83D has been shown to regulate the expression of cancer stem cell markers such as CD44 and its variants, suggesting a role in maintaining cancer stemness .

What are the optimal applications for biotin-conjugated FAM83D antibodies versus unconjugated versions?

Biotin-conjugated FAM83D antibodies offer distinct advantages for specific research applications compared to unconjugated versions. The primary benefit lies in the strong, specific binding between biotin and streptavidin/avidin, which creates versatile detection systems with amplification capabilities .

For ELISA applications, biotin-conjugated FAM83D antibodies (such as those targeting AA 339-470) provide enhanced sensitivity through signal amplification when paired with streptavidin-HRP or streptavidin-fluorophore conjugates . This makes them particularly useful for detecting low-abundance FAM83D in complex biological samples.

In immunoprecipitation studies, biotin-conjugated antibodies enable efficient pull-down of FAM83D and associated proteins using streptavidin-coated beads, allowing for clean isolation of protein complexes for downstream analysis. This is particularly valuable for studying mitosis-specific interactions between FAM83D and CK1α .

For immunohistochemistry and immunofluorescence applications, biotin-conjugated antibodies facilitate multi-layered staining approaches and signal amplification systems, which can be critical when examining FAM83D expression in tissue microarrays or cellular localization during mitosis .

What validation methods should be employed to confirm FAM83D antibody specificity?

Comprehensive validation of biotin-conjugated FAM83D antibody specificity requires multiple complementary approaches:

• Western Blot Validation: Compare antibody reactivity in FAM83D-expressing versus FAM83D-knockout cell lines. A specific antibody should detect bands at the expected molecular weight in wild-type cells but show no signal in knockout lines . Observe for potential cross-reactivity with other FAM83 family members.

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (AA 339-470) before application in Western blot or immunostaining. Signal elimination confirms specific binding to the target epitope .

• RNA Interference Correlation: Perform siRNA or shRNA knockdown of FAM83D and confirm that antibody signal diminishes proportionally to knockdown efficiency using quantitative Western blot or immunofluorescence .

• Immunoprecipitation-Mass Spectrometry: Use the antibody for immunoprecipitation followed by mass spectrometry to confirm that FAM83D is the predominant protein pulled down .

• Subcellular Localization Assessment: Confirm that immunostaining patterns match the expected subcellular distribution of FAM83D, particularly its mitotic spindle association during cell division .

• Multiple Antibody Concordance: Compare staining patterns using multiple antibodies targeting different FAM83D epitopes to ensure consistent results.

• Correlation with mRNA Expression: Analyze whether antibody signal intensity correlates with FAM83D mRNA levels across cell lines with varying expression levels .

What experimental parameters are critical for successful immunoprecipitation of FAM83D-protein complexes?

Successful immunoprecipitation of FAM83D-protein complexes with biotin-conjugated antibodies requires careful optimization of several parameters:

• Cell Synchronization: For studying interactions such as FAM83D-CK1α, synchronize cells in mitosis using nocodazole or STLC, as these interactions are cell cycle-dependent . Flow cytometry with propidium iodide staining can confirm mitotic enrichment.

• Lysis Buffer Composition: Use buffers containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40 or 1% Triton X-100, with protease and phosphatase inhibitors to preserve protein-protein interactions .

• Antibody Immobilization: For biotin-conjugated antibodies, pre-bind to streptavidin-coated magnetic beads at a ratio of approximately 5-10 μg antibody per 50 μl bead slurry for maximum capture efficiency.

• Input Protein Concentration: Use sufficient protein input (500-1000 μg total protein) while maintaining antibody excess to ensure complete capture of target complexes.

• Incubation Conditions: Perform binding at 4°C for 3-4 hours or overnight with gentle rotation to maintain complex integrity while allowing sufficient binding time.

• Washing Stringency: Balance between preserving specific interactions and removing non-specific binding through sequential washes of decreasing stringency (high salt to low salt).

• Elution Method: For biotin-conjugated antibodies on streptavidin beads, direct elution in SDS sample buffer at 95°C is effective, though gentler methods may better preserve complex integrity.

• Controls: Include IgG control immunoprecipitations and lysates from FAM83D-knockout cells to identify non-specific binding .

Research has demonstrated that these parameters are critical for successfully isolating the FAM83D-CK1α complex, which forms specifically during mitosis and is essential for proper spindle positioning .

How should researchers optimize detection conditions for FAM83D in different cellular contexts?

Optimizing FAM83D detection across different cellular contexts requires tailored approaches based on expression levels and experimental goals:

• Expression Level Assessment: First determine baseline FAM83D expression through qRT-PCR across cell lines, as expression varies significantly between non-malignant cells (like MCF10A) and cancer cell lines, which often show 1.5-4 fold higher expression .

• Western Blot Detection:

  • For high-expressing cells: Use antibody dilutions of 1:1000-1:2000

  • For low-expressing cells: Increase protein loading (50-100 μg) and employ enhanced chemiluminescence detection systems

  • Use 7.5-10% gels for optimal resolution of FAM83D (~60 kDa)

  • Note the mitotic electrophoretic mobility shift when analyzing synchronized cells

• Immunofluorescence Optimization:

  • Fixation method: 4% paraformaldehyde (10 min) for structural preservation or methanol (-20°C, 10 min) for enhanced epitope accessibility

  • Permeabilization: 0.1-0.5% Triton X-100 for 10 minutes

  • Blocking: 5% BSA or normal serum from secondary antibody species

  • For mitotic spindle visualization: Co-stain with tubulin and DNA markers

  • For low expression: Implement biotin-streptavidin amplification systems

• Flow Cytometry Protocol Refinement:

  • For total FAM83D: Standard fixation/permeabilization protocols

  • For detecting changes in CD44 expression following FAM83D modulation: Dual staining with appropriate compensation controls

  • Double staining with cell cycle markers to correlate FAM83D levels with cell cycle phase

• ELISA Detection Systems:

  • For biotin-conjugated antibodies: Use streptavidin-HRP at 1:1000-1:5000

  • Standard curve generation: Use recombinant FAM83D protein (AA 339-470)

  • Sample preparation: Extract proteins with RIPA buffer containing protease inhibitors

These optimizations enable reliable detection of FAM83D across experimental contexts, from basic expression analysis to complex co-localization and interaction studies.

How can FAM83D antibodies be utilized to investigate its role in mitotic spindle organization?

To investigate FAM83D's role in mitotic spindle organization using biotin-conjugated antibodies, researchers should implement a multi-faceted experimental approach:

• High-Resolution Co-localization Analysis:

  • Synchronize cells in mitosis using STLC or nocodazole treatment

  • Perform triple immunofluorescence with biotin-FAM83D antibody (detected via streptavidin-fluorophore), α-tubulin (spindle marker), and γ-tubulin (centrosome marker)

  • Use confocal or super-resolution microscopy to precisely map FAM83D localization during different mitotic phases

  • Quantify co-localization coefficients between FAM83D and spindle components

• Spindle Recruitment Dynamics:

  • Combine immunofluorescence with time-lapse imaging in synchronized cells

  • Correlate FAM83D spindle association with CK1α recruitment timing

  • Implement FRAP (Fluorescence Recovery After Photobleaching) using fluorescently-tagged FAM83D to measure spindle binding dynamics

• Protein-Protein Interaction Network Analysis:

  • Perform spindle isolation followed by immunoprecipitation with biotin-FAM83D antibody

  • Identify mitotic spindle interaction partners through mass spectrometry

  • Validate key interactions using proximity ligation assay (PLA) between FAM83D and candidate proteins

• Functional Manipulation Studies:

  • Compare spindle morphology and function in wild-type versus FAM83D-knockout cells

  • Rescue experiments with wild-type FAM83D versus CK1α-binding deficient mutants

  • Live-cell imaging to measure mitotic progression timing and spindle positioning

• Phosphorylation Profiling:

  • Identify CK1α-dependent phosphorylation events on spindle proteins in the presence/absence of FAM83D

  • Use phospho-specific antibodies to monitor key regulatory phosphorylation sites

Research has demonstrated that FAM83D localizes to the spindle and recruits CK1α during mitosis, with cells lacking FAM83D or expressing CK1α-binding-deficient FAM83D mutants showing improper spindle positioning and delayed progression through mitosis .

What methodologies are most effective for studying the relationship between FAM83D and CK1α?

The FAM83D-CK1α interaction represents a critical regulatory mechanism in mitotic progression. To effectively study this relationship, researchers should employ these methodological approaches:

• Temporal Analysis of Complex Formation:

  • Synchronize cells at different cell cycle stages (G1, S, G2, prometaphase)

  • Perform co-immunoprecipitation with biotin-FAM83D antibody at each stage

  • Quantify relative amounts of CK1α association by immunoblotting

  • This approach has revealed that the interaction is specifically enhanced during mitosis

• Domain Mapping and Mutational Analysis:

  • Generate FAM83D constructs with mutations in the DUF1669 domain, particularly at F284A/F288A positions critical for CK1 binding

  • Compare wild-type versus mutant FAM83D ability to co-precipitate CK1α

  • Use purified recombinant proteins for in vitro binding assays to determine binding affinities

• Subcellular Co-localization Dynamics:

  • Implement proximity ligation assay (PLA) using biotin-FAM83D antibody and CK1α antibody

  • Quantify PLA signals throughout mitotic progression

  • Correlate spatial distribution of the complex with spindle dynamics

• Functional Consequence Assessment:

  • Compare mitotic progression timing in cells expressing wild-type FAM83D versus CK1α-binding deficient mutants

  • Measure spindle positioning defects using live-cell imaging

  • Analyze chromosome segregation errors resulting from disrupted FAM83D-CK1α interaction

• CK1α Substrate Identification in FAM83D-dependent Context:

  • Perform phosphoproteomic analysis comparing wild-type versus FAM83D-knockout cells

  • Identify CK1α-dependent phosphorylation events that require FAM83D scaffolding

  • Validate key substrates using phospho-specific antibodies

• Competitive Inhibition Studies:

  • Use peptides derived from the CK1α-binding region of FAM83D as competitive inhibitors

  • Assess disruption of endogenous complex formation

  • Measure functional consequences on mitotic progression

Research has demonstrated that FAM83D facilitates CK1α recruitment to the mitotic spindle through direct interaction, and disruption of this interaction leads to spindle positioning defects and mitotic delay .

How can FAM83D expression analysis be integrated into cancer progression assessment?

Integrating FAM83D expression analysis into cancer progression assessment requires a comprehensive approach spanning from molecular characterization to clinical correlation:

• Expression Correlation with Clinicopathological Parameters:

  • Perform immunohistochemistry on tissue microarrays using biotin-conjugated FAM83D antibodies

  • Quantify staining intensity across tumor stages (I-IV)

  • Correlate expression with tumor grade, lymph node invasion, and distant metastasis

  • Research has demonstrated that high FAM83D expression significantly associates with advanced clinical stage and higher tumor grade in HCC patients

• Multiparameter Flow Cytometry Analysis:

  • Develop panels combining FAM83D with established cancer progression markers

  • Include markers for proliferation (Ki-67), stemness (CD44), and epithelial-mesenchymal transition

  • Apply to circulating tumor cells or fine-needle aspirates

  • Studies show FAM83D knockdown reduces CD44 expression in cancer cells

• Longitudinal Monitoring Approaches:

  • Serial liquid biopsies with CTCs analyzed for FAM83D expression

  • Before/after treatment comparisons to assess therapy response

  • Integration with circulating tumor DNA analysis

• Multiplexed Biomarker Assessment:

  • Combine FAM83D with established prognostic markers

  • Create prediction models incorporating multiple parameters

  • Validate prognostic value in independent patient cohorts

  • Research shows high FAM83D expression correlates with shortened distant metastasis-free survival in breast cancer patients

• Functional Pathway Association:

  • Correlate FAM83D expression with migration/invasion protein levels

  • Analyze association with apoptosis regulators (Bcl-2, Bax, PARP)

  • Studies demonstrate FAM83D knockdown reduces migration protein levels and increases apoptotic markers

This integrated approach enables comprehensive assessment of FAM83D's role in cancer progression across different cancer types and treatment contexts.

What are the optimal experimental designs for investigating FAM83D's influence on cell migration and invasion?

To comprehensively investigate FAM83D's influence on cell migration and invasion, researchers should implement a systematic experimental design combining functional assays with molecular analyses:

• 2D Migration Assays:

  • Scratch Wound Healing:

    • Create standardized wounds in FAM83D-modulated cell monolayers

    • Capture time-lapse images over 24-48 hours

    • Quantify wound closure rates using automated image analysis

    • Research shows FAM83D overexpression accelerates wound closure while knockdown reduces closure rate

  • Single Cell Tracking:

    • Monitor individual cell trajectories in sparse cultures

    • Calculate velocity, directionality, and persistence

    • Compare FAM83D wild-type, knockdown, and overexpression conditions

• 3D Invasion Assays:

  • Boyden Chamber/Transwell Assays:

    • Uncoated membranes for migration assessment

    • Matrigel-coated membranes for invasion potential

    • Quantify cells traversing membrane after 24-48 hours

    • Studies demonstrate FAM83D overexpression increases both migration and invasion capacity

  • 3D Spheroid Invasion:

    • Generate spheroids from FAM83D-modulated cells

    • Embed in Matrigel or collagen matrices

    • Measure invasion distance and pattern over 1-7 days

• Molecular Mechanism Analysis:

  • Migration Protein Expression Profiling:

    • Analyze key regulators (focal adhesion proteins, Rho GTPases)

    • Compare expression levels by Western blot after FAM83D modulation

    • Research shows FAM83D knockdown significantly downregulates migration-associated proteins

  • EMT Marker Assessment:

    • Monitor E-cadherin, N-cadherin, vimentin expression

    • Evaluate transcription factors (Snail, Slug, ZEB1/2)

    • Correlate with FAM83D expression levels

• Live-Cell Cytoskeletal Dynamics:

  • Focal Adhesion Turnover:

    • Transfect cells with fluorescent focal adhesion markers

    • Measure assembly/disassembly rates after FAM83D modulation

    • Calculate adhesion lifetimes and distribution

  • Actin Cytoskeleton Remodeling:

    • Visualize F-actin using fluorescent phalloidin

    • Quantify stress fiber formation and lamellipodia dynamics

    • Correlate with migration capacity

• Rescue Experiments:

  • Perform FAM83D knockdown followed by re-expression of:

    • Wild-type FAM83D

    • CK1α-binding deficient mutants

    • FBXW7-interaction domain mutants

  • Assess which domains are essential for migration/invasion phenotypes

These approaches collectively provide a comprehensive assessment of FAM83D's role in promoting cell migration and invasion, key processes in cancer metastasis.

How can researchers investigate the mechanisms by which FAM83D regulates apoptosis in cancer cells?

To investigate FAM83D's role in apoptosis regulation, researchers should implement a comprehensive experimental strategy combining functional assays with molecular pathway analysis:

• Quantitative Apoptosis Assessment:

  • Flow Cytometry Analysis:

    • Annexin V-PE/7-AAD double staining to distinguish early/late apoptosis

    • Compare FAM83D knockdown versus control conditions

    • Research demonstrates FAM83D knockdown promotes early apoptosis in HCC cell lines

  • Caspase Activity Assays:

    • Measure caspase-3/7, caspase-8, and caspase-9 activities

    • Determine whether FAM83D affects intrinsic or extrinsic pathways

    • Correlate activity levels with FAM83D expression

• Apoptotic Protein Expression Analysis:

  • Western Blot Profiling:

    • Monitor key regulators: Bcl-2 (anti-apoptotic), Bax (pro-apoptotic), PARP

    • Compare expression levels after FAM83D modulation

    • Studies show FAM83D knockdown reduces Bcl-2 while increasing Bax and PARP

  • Subcellular Fractionation:

    • Assess mitochondrial translocation of Bax

    • Measure cytochrome c release into cytosol

    • Determine if FAM83D affects mitochondrial membrane integrity

• Mechanistic Pathway Investigation:

  • FBXW7 Interaction Studies:

    • Evaluate whether FAM83D-FBXW7 interaction affects apoptotic regulation

    • Determine if FBXW7 substrates involved in apoptosis are regulated by FAM83D

    • Research shows FAM83D physically interacts with FBXW7, a tumor suppressor

  • Kinase Signaling Analysis:

    • Assess activation status of survival pathways (PI3K/AKT, MAPK)

    • Determine if FAM83D modulates these pathways to regulate apoptosis

    • Investigate potential CK1α-dependent mechanisms

• Apoptotic Stimulus Response:

  • Dose-Response Studies:

    • Challenge cells with apoptotic inducers (staurosporine, TRAIL, cisplatin)

    • Compare sensitivity in FAM83D wild-type versus knockdown cells

    • Determine if FAM83D confers resistance to specific apoptotic stimuli

  • Recovery Assays:

    • Measure cell survival after transient apoptotic stimulus

    • Assess whether FAM83D affects recovery capacity

• Rescue Experiments:

  • Domain Function Analysis:

    • Express FAM83D mutants lacking specific functional domains

    • Determine which domains are essential for apoptosis regulation

    • Assess whether CK1α binding is required for anti-apoptotic function

  • Downstream Effector Modulation:

    • Combine FAM83D knockdown with Bcl-2 overexpression

    • Determine if apoptotic phenotype can be rescued

The table below summarizes key apoptotic markers affected by FAM83D modulation based on published research:

These methodologies provide a comprehensive framework for elucidating the molecular mechanisms through which FAM83D regulates apoptosis in cancer cells.