FAM49B Antibody

What is FAM49B and what cellular functions does it regulate?

FAM49B (Family with sequence similarity 49 member B) is a 324-amino acid protein (36.7 kDa) that functions as a CYFIP-related Rac1 interactor B, encoded by the CYRIB gene . It plays critical roles in regulating cellular processes including apoptosis, cell proliferation, and migration. FAM49B is primarily localized in the mitochondria and membrane, and is widely expressed across multiple tissues including the cerebral cortex and kidney . As a member of the CYRI protein family, FAM49B has been identified as a potential tumor suppressor, making it significant in cancer research where it may serve as a therapeutic target .

What are the key applications for FAM49B antibodies in research?

FAM49B antibodies have been validated for several laboratory applications that enable researchers to study this protein's expression and function:

The D-8 monoclonal antibody, for example, detects FAM49B across multiple species including human, mouse, and rat models .

How should I select the appropriate FAM49B antibody for my experiment?

When selecting a FAM49B antibody, consider these key factors:

• Experimental application: Different antibodies are optimized for specific techniques (WB, IF, IHC, etc.)

• Species reactivity: Verify the antibody detects FAM49B in your experimental model organism

• Clonality: Monoclonal antibodies (like D-8) offer high specificity for a single epitope, while polyclonal antibodies (like PACO41174) recognize multiple epitopes

• Conjugation needs: Determine if your experiment requires unconjugated antibody or specific conjugations (HRP, FITC, PE, etc.)

• Validation evidence: Review published citations where available to confirm performance in similar experimental contexts

For new research projects, consider testing multiple antibodies in parallel to determine which performs best in your specific experimental system.

How does FAM49B expression correlate with cancer progression?

FAM49B has been implicated in cancer development through its role in regulating cell proliferation, apoptosis, and migration . Research suggests that its gene location on human chromosome 8q24.21 is particularly significant, as this region has been implicated in several genetic disorders and cancers . Notably, alterations in chromosome 8, such as translocations and amplifications of the c-Myc gene, correlate with poor prognoses in leukemias and lymphomas .

When designing studies to investigate this correlation, researchers should:

• Compare FAM49B expression levels across cancer progression stages using quantitative Western blotting

• Perform IHC studies on tissue microarrays to evaluate expression patterns in large patient cohorts

• Correlate expression data with clinical outcomes to establish prognostic significance

• Investigate the functional impact of FAM49B knockdown or overexpression on cancer cell phenotypes

What are the methodological considerations when studying the two alternative splice isoforms of FAM49B?

FAM49B exists in two alternatively spliced isoforms which may exhibit different functional properties and regulatory mechanisms . When investigating these isoforms:

• Select antibodies that can differentiate between both isoforms, or choose isoform-specific antibodies when available

• Use RT-PCR with isoform-specific primers to confirm expression at the mRNA level

• Design experimental controls that can distinguish functional differences between isoforms

• Consider the tissue-specific expression patterns of each isoform

• When performing knockdown experiments, ensure your approach targets the relevant isoform(s)

The methodological challenge lies in distinguishing these isoforms' unique contributions to cellular processes, as their functional differences remain incompletely characterized.

How can I effectively study the interaction between FAM49B and the Rac1 signaling pathway?

As CYRIB (FAM49B) functions as a Rac1 interactor , researchers investigating this pathway should:

• Use co-immunoprecipitation with FAM49B antibodies to pull down protein complexes, followed by Western blotting for Rac1 and other pathway components

• Perform proximity ligation assays to visualize FAM49B-Rac1 interactions in situ

• Design FRET or BiFC assays to monitor dynamic interactions

• Use Rac1 activity assays in conjunction with FAM49B modulation (overexpression/knockdown) to assess functional impacts

• Consider the subcellular localization of these interactions using fractionation approaches followed by immunoblotting

This multi-method approach provides more robust evidence of interaction than any single technique alone.

What are the optimal conditions for Western blotting with FAM49B antibodies?

Western blotting for FAM49B requires careful optimization:

For optimal results, include positive controls (tissues/cells known to express FAM49B) and consider these technical notes:

• Both reducing and non-reducing conditions may be tested

• For challenging samples, immunoprecipitation followed by Western blotting may increase sensitivity

• When studying both isoforms, use gradient gels (4-15%) to better resolve the size differences

How should I approach FAM49B antibody validation for my specific experimental system?

Proper validation ensures reliable results and should include:

• Positive and negative control samples (tissues/cells with known expression patterns)

• Peptide competition assays to confirm specificity

• Knockdown/knockout validation using siRNA or CRISPR-based approaches

• Cross-validation with multiple antibodies targeting different epitopes

• Comparison of results across multiple detection techniques

For human samples, the FAM49B Polyclonal Antibody (PAC041174) has been validated for Western blot applications , while the D-8 monoclonal antibody provides multi-species reactivity across human, mouse, and rat samples .

What considerations are important when using FAM49B antibodies for immunofluorescence and immunohistochemistry?

For optimal imaging applications:

• Fixation method: Test both paraformaldehyde (4%) and methanol fixation, as epitope accessibility may differ

• Antigen retrieval: For FFPE tissues, citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be compared

• Blocking: Use species-appropriate serum (5-10%) with 0.1-0.3% Triton X-100 for permeabilization

• Antibody incubation: Overnight at 4°C typically yields best results at dilutions of 1:50-1:200

• Controls: Include no-primary controls and positive control tissues

When co-staining with other markers, carefully select secondary antibodies to avoid cross-reactivity and consider chromogenic detection for tissues with high autofluorescence.

How can I address inconsistent FAM49B antibody staining patterns in different tissue types?

Inconsistent staining may result from several factors:

• Tissue-specific expression levels: FAM49B shows variable expression across tissues, with notable presence in cerebral cortex and kidney

• Isoform variation: The two alternatively spliced isoforms may exhibit different tissue distributions

• Epitope masking: Protein-protein interactions or post-translational modifications may obscure antibody binding sites

• Fixation sensitivity: Some epitopes are particularly sensitive to fixation method and duration

Methodological solutions include:

• Testing multiple fixation protocols

• Comparing multiple antibodies targeting different epitopes

• Using fresh-frozen tissues alongside FFPE samples

• Validating with mRNA expression (ISH or RT-PCR) from the same tissue regions

What explains the discrepancy in molecular weight detection between expected (36.7 kDa) and observed (39.1 kDa) FAM49B protein?

This common discrepancy has several potential explanations:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications increase molecular weight

• Isoform detection: The antibody may be detecting the larger splice variant

• SDS-PAGE anomalies: Some proteins migrate abnormally due to their amino acid composition

• Fusion tags: In recombinant systems, fusion tags contribute additional mass

To address this:

• Use mass spectrometry to confirm the exact protein being detected

• Examine the amino acid sequence for post-translational modification sites

• Compare migration patterns after phosphatase treatment

• Run parallel samples from different tissue sources to identify tissue-specific modifications

How should I interpret contradictory results between FAM49B detection methods?

When facing contradicting results:

• Consider method-specific limitations:

  • Western blot provides protein size but may detect denatured epitopes

  • IHC/IF provides localization but may suffer from cross-reactivity

  • ELISA offers quantification but may detect both free and complexed protein

• Methodological approach to resolution:

  • Validate with orthogonal techniques (e.g., mass spectrometry)

  • Use genetic approaches (knockdown/overexpression) to confirm specificity

  • Consider splice variants and post-translational modifications

  • Review subcellular fractionation to confirm localization patterns

  • Test multiple antibodies targeting different epitopes

How does FAM49B relate to neuropsychiatric disorders associated with chromosome 8?

Research has linked chromosome 8, where the FAM49B gene is located (8q24.21), to psychiatric conditions including schizophrenia and bipolar disorder . When investigating this connection:

• Consider using FAM49B antibodies in post-mortem brain tissue studies

• Compare expression levels between patient and control samples

• Investigate genetic variations (SNPs) in the FAM49B gene in neuropsychiatric cohorts

• Explore FAM49B's role in neuronal development and function using primary neuron cultures

• Examine potential interactions with other psychiatric risk genes

This emerging area represents an opportunity to expand FAM49B research beyond cancer into neuroscience applications.

What experimental approaches can assess FAM49B as a potential therapeutic target?

As FAM49B has been implicated as a potential therapeutic target for cancer treatment and prevention , researchers should consider:

• High-throughput screening approaches:

  • siRNA/CRISPR screens to identify synthetic lethal interactions

  • Small molecule screens targeting FAM49B function or expression

  • Peptide inhibitors of protein-protein interactions

• Preclinical validation studies:

  • Genetic modulation in xenograft models

  • Correlation of expression with treatment response

  • Combination approaches with standard chemotherapeutics

• Biomarker development:

  • IHC optimization for potential diagnostic applications

  • Circulating protein detection methods

  • Correlation with disease progression and treatment response