FAM50B Antibody, Biotin conjugated

What is FAM50B and what is its biological significance?

FAM50B (Family with Sequence Similarity 50, Member B) is a protein containing an intronless ORF that arose from ancestral retroposition. It is related to a plant protein involved in the circadian clock. The gene is imprinted and paternally expressed in many tissues, located adjacent to a differentially methylated region (DMR) . FAM50B plays important roles in cellular processes including cell division and proliferation. Research has identified FAM50B as a paralogue of FAM50A, with their interaction being particularly significant in cancer research .

How does FAM50B differ from its paralogue FAM50A?

FAM50A and FAM50B are paralogues with related but distinct functions:

FAM50A/FAM50B have been identified as a synthetic lethal pair, where disruption of FAM50A in cells with silenced FAM50B leads to loss of cellular fitness .

What applications are FAM50B antibodies most commonly used for?

FAM50B antibodies are valuable tools in various experimental techniques:

• Immunohistochemistry (IHC): Detection in paraffin-embedded tissues

• Immunofluorescence (IF): Subcellular localization studies

• ELISA: Quantitative protein detection

• Western blotting: Protein expression analysis

• Antibody production: As immunogens for developing new antibodies

Biotin-conjugated antibodies offer additional advantages for detection sensitivity through the strong biotin-streptavidin interaction.

What is the typical subcellular localization pattern observed with FAM50B antibodies?

Based on information about its paralogue FAM50A, which shows nuclear localization , FAM50B is likely to demonstrate nuclear staining patterns. When performing immunofluorescence or immunohistochemistry, researchers should expect to observe nuclear signals, particularly if FAM50B functions in transcriptional regulation similar to FAM50A. Validation studies with FAM50B antibodies have been performed in human brain tissue, confirming its expression pattern in neural cells .

What are recommended dilutions for FAM50B antibodies in various applications?

While specific recommendations for biotin-conjugated FAM50B antibodies vary by manufacturer, general guidelines based on similar antibodies suggest:

Always perform a dilution series with your specific samples to determine optimal concentrations.

What controls should I include when using FAM50B antibodies?

For rigorous validation of FAM50B antibody experiments:

• Positive control: Cell lines or tissues known to express FAM50B

• Negative control: Samples with FAM50B knockdown/knockout or tissues known not to express FAM50B

• Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific staining

• Isotype control: Matching concentration of non-specific IgG from same host species

• Secondary-only control: Omit primary antibody to assess non-specific binding

• Endogenous biotin control: For biotin-conjugated antibodies, include streptavidin-only control to assess endogenous biotin

How should I prepare samples for optimal FAM50B detection?

For nuclear proteins like FAM50B:

• Fixation: 4% paraformaldehyde (15-20 minutes) for cells; formalin-fixed paraffin-embedded for tissues

• Antigen retrieval: For FFPE tissues, high-pressure citrate buffer (pH 6.0) treatment is effective

• Permeabilization: Use 0.1-0.5% Triton X-100 to ensure nuclear access

• Blocking: 10% normal goat serum for 30 minutes at room temperature

• For biotin-conjugated antibodies: Include avidin/biotin blocking step to reduce background

• Incubation: Optimal conditions depend on application, but overnight at 4°C often yields best results for primary antibodies

How can I investigate the synthetic lethal relationship between FAM50A and FAM50B?

The synthetic lethality between FAM50A and FAM50B provides an important research avenue:

• Verification: Confirm FAM50B silencing/expression in your cancer model using antibodies

• Cellular fitness assays: Measure proliferation, apoptosis, and cell cycle effects after FAM50A disruption in FAM50B-silenced cells

• Rescue experiments: Re-express FAM50B to confirm specificity of the synthetic lethal effect

• Mechanistic studies: Analyze transcriptional perturbations and micronucleus formation reported in FAM50A/FAM50B disrupted cells

• Clinical correlation: Screen patient samples for FAM50B silencing status using antibodies (occurs in ~4% of cancers)

• Therapeutic development: Test FAM50A-targeting approaches in FAM50B-silent cancer models

What approaches can I use to study FAM50B's role in transcriptional regulation?

Since FAM50B disruption causes extensive perturbation of transcriptional programs , and its paralogue FAM50A may function as a DNA-binding protein or transcription factor :

• ChIP experiments: Use FAM50B antibodies to identify genomic binding sites

• Co-immunoprecipitation: Identify protein interaction partners in transcriptional complexes

• RNA-seq following manipulation: Analyze transcriptional changes after FAM50B knockdown/overexpression

• Nuclear/cytoplasmic fractionation: Confirm nuclear localization and potential shuttling

• Immunofluorescence co-localization: Examine spatial relationships with known transcription factors

• Epigenetic analysis: Investigate relationships with chromatin modifications at binding sites

How can I optimize immunoprecipitation protocols using biotin-conjugated FAM50B antibodies?

For effective immunoprecipitation:

• Nuclear extraction: Use appropriate nuclear lysis buffers with 0.1-0.5% NP-40 or Triton X-100

• Sonication: Consider brief sonication to disrupt nuclear membranes while preserving protein integrity

• Pre-clearing: Use streptavidin beads to reduce non-specific binding

• Antibody binding: For biotin-conjugated antibodies, use streptavidin-coated magnetic beads for capture

• Washing conditions: Optimize stringency to maintain specific interactions while reducing background

• Elution: Use biotin competition or denaturing conditions depending on downstream applications

• Controls: Include IgG control and input samples for accurate normalization

What are common issues when using biotin-conjugated antibodies and how can I address them?

How do I interpret contradictory results between FAM50B protein detection and transcriptional data?

When faced with discrepancies:

• Imprinting effects: As an imprinted gene, FAM50B expression is regulated by parent-of-origin effects, potentially causing heterogeneous expression

• Post-transcriptional regulation: Consider miRNA or RNA-binding protein influences

• Protein stability: Analyze protein half-life under different conditions

• Antibody epitope accessibility: Post-translational modifications might mask epitopes

• Technical validation: Use multiple antibodies targeting different epitopes

• Spatial/temporal factors: FAM50B expression may vary across cell cycle or differentiation states

How can I validate the specificity of my FAM50B antibody?

To ensure antibody specificity:

• Peptide competition: Pre-incubation with the immunizing peptide should abolish specific signal

• Knockdown/knockout validation: Compare staining in FAM50B-depleted vs. control cells

• Recombinant protein: Test against purified FAM50B protein (e.g., ABIN2720793 )

• Orthogonal methods: Confirm findings using alternative detection methods

• Cross-reactivity testing: Verify absence of signal in tissues lacking FAM50B expression

• Multiple antibodies: Use antibodies recognizing different epitopes to confirm results

What storage and handling conditions are optimal for maintaining FAM50B antibody activity?

For maximum antibody stability and performance:

• Storage temperature: Store at -20°C for optimal long-term stability

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles

• Buffer composition: Typically stored in buffered aqueous glycerol solutions

• Working dilutions: Prepare fresh and use within 24 hours

• Transportation: Ship on wet ice to maintain antibody integrity

• Long-term stability: Most antibodies remain stable for 12 months when properly stored

How does the biotin conjugation affect experimental design compared to unconjugated antibodies?

Biotin conjugation introduces specific considerations:

• Detection systems: Requires streptavidin-conjugated reporter molecules rather than species-specific secondary antibodies

• Signal amplification: Offers enhanced sensitivity through multiple streptavidin binding

• Endogenous biotin: Requires additional blocking steps to reduce background

• Multiplexing: Enables simultaneous detection with antibodies from the same host species

• Direct detection: Eliminates secondary antibody cross-reactivity concerns

• Stability: May have different storage requirements than unconjugated versions

What are the considerations for using FAM50B antibodies in co-immunostaining experiments?

For successful multiple labeling:

• Antibody compatibility: Select primary antibodies from different host species when using non-conjugated antibodies

• Sequential staining: Consider sequential rather than simultaneous incubation for biotin-conjugated antibodies

• Channel selection: Choose fluorophores with minimal spectral overlap

• Controls: Include single-stained controls to assess bleed-through

• Order of application: Apply the weakest signal antibody first for best results

• Blocking steps: Include additional blocking between sequential applications

How might understanding FAM50B function contribute to cancer therapeutics?

The FAM50A/FAM50B synthetic lethal relationship offers translational potential:

• Biomarker development: Screening tumors for FAM50B silencing could identify candidates for FAM50A-targeted therapy

• Drug development: FAM50A inhibitors could be developed for treating FAM50B-silent tumors

• Combination approaches: FAM50A targeting might sensitize cells to existing therapies

• Resistance mechanisms: Understanding how cells overcome the synthetic lethality could reveal additional targets

• Transcriptional dependencies: The widespread transcriptional effects of FAM50B loss suggest potential vulnerability to transcription-targeting drugs

• Cell cycle targeting: Given FAM50B's role in cell division, combining with cell cycle inhibitors might be synergistic

What emerging technologies might enhance our understanding of FAM50B function?

Cutting-edge approaches offer new insights:

• Single-cell technologies: Reveal cell-specific expression patterns and effects

• Spatial transcriptomics: Map FAM50B expression within tissue architecture

• CRISPR screening: Identify additional synthetic lethal partners

• Proteomics: Define the FAM50B interactome under various conditions

• Cryo-EM: Determine structural details of FAM50B-containing complexes

• Live-cell imaging: Monitor dynamic FAM50B localization and interactions

How might epigenetic regulation influence FAM50B antibody-based detection?

Considering FAM50B's imprinted status :

• Methylation interference: DNA methylation might affect chromatin structure and epitope accessibility

• Tissue-specific expression: Imprinting can lead to tissue-specific expression patterns requiring validation

• Developmental regulation: Expression may change during development and differentiation

• Pathological changes: Disease states might alter epigenetic regulation of FAM50B

• Cell line validation: Cell lines may not maintain proper imprinting status

• Technical approaches: Combining antibody detection with methylation analysis could provide mechanistic insights