FAM149B1 Antibody

What is FAM149B1 and why is it important in research?

FAM149B1 (Family with Sequence Similarity 149, Member B1) is involved in the localization of proteins to the cilium and cilium assembly. It indirectly regulates the signaling functions of the cilium, being required for normal SHH/smoothened signaling and proper development . Its significance stems from its association with Joubert Syndrome 36 and Orofaciodigital Syndrome VI, ciliopathies characterized by neurological, skeletal, and oculomotor abnormalities . Biallelic truncating mutations in FAM149B1 result in cilia dysfunction , making it an important target for researchers studying ciliopathies and developmental disorders.

FAM149B1 antibodies have been validated for multiple applications with specific methodological considerations:

• Western Blotting (WB): Recommended dilutions range from 0.04-0.4 μg/ml to 1:300. The expected band size is approximately 65 kDa .

• Immunohistochemistry (IHC): Dilutions typically range from 1:20-1:500, with HIER pH 6 retrieval recommended for paraffin-embedded tissues .

• Immunofluorescence (IF): For cultured cells and paraffin sections, using PFA/Triton X-100 fixation/permeabilization is recommended .

• ELISA: Several antibodies are validated for ELISA applications, though specific protocols vary by manufacturer .

How should I select the appropriate FAM149B1 antibody epitope for my specific research questions?

Selection should be guided by your research objectives and the structural/functional domains of FAM149B1:

• For studying full-length protein: Choose antibodies targeting AA 107-528, which covers most of the protein sequence .

• For DUF3719 domain studies: This domain is critical for FAM149B1 function. Select antibodies targeting regions containing this domain (approximately AA 150-300) .

• For N-terminal studies: Antibodies targeting AA 21-120 are suitable, especially for distinguishing between potential truncated variants .

• For interaction studies with TBC1D32: Since FAM149B1 interacts with TBC1D32, epitopes away from the interaction interface should be selected to avoid interference .

When studying disease-associated mutations, avoid antibodies targeting regions that may be affected by the mutations (e.g., the p.Gln118Hisfs*20 variant would affect epitopes beyond AA 118) .

What controls should be included when working with FAM149B1 antibodies?

Rigorous control strategies should include:

• Positive tissue controls: Human testis and spleen show strong cytoplasmic positivity .

• Negative controls: Include samples known to lack FAM149B1 expression.

• Peptide competition assays: Pre-incubate the antibody with the immunogen peptide to confirm specificity.

• Genetic controls: When possible, use FAM149B1 knockout or siRNA knockdown cells as negative controls .

• Cross-reactivity checks: If working with non-human samples, verify species cross-reactivity, noting that mouse orthologs share 68-81% sequence identity with human targets, depending on the epitope region .

What are the optimal sample preparation methods for detecting FAM149B1 in ciliary structures?

For successful detection of FAM149B1 in ciliary structures:

• Cell culture conditions: Induce ciliogenesis by serum starvation for 24-48 hours before fixation .

• Fixation protocol:

  • For immunofluorescence: Use 4% PFA for 10 minutes at room temperature

  • For electron microscopy: Use 2.5% glutaraldehyde followed by 1% osmium tetroxide

• Permeabilization: Use 0.1% Triton X-100 for 5-10 minutes (avoid harsher detergents that may disrupt ciliary structure) .

• Co-staining markers: Include established ciliary markers (acetylated tubulin, ARL13B, or IFT88) to confirm ciliary localization .

• Imaging considerations: Use confocal microscopy with z-stacking to properly visualize the three-dimensional structure of cilia.

How can FAM149B1 antibodies be used to investigate ciliopathy pathogenesis mechanisms?

To investigate ciliopathy mechanisms using FAM149B1 antibodies:

• Ciliary morphology analysis: FAM149B1-deficient cells display abnormal morphology with bulbous ciliary tips. Use immunostaining to quantify:

  • Ciliary length (increased in FAM149B1 mutants: 3.9μm vs 2.7-2.9μm in controls)

  • Tip morphology (bulbous appearance in mutants)

  • IFT protein accumulation at distal tips

• SHH signaling assessment: FAM149B1 is required for normal SHH signaling. Design experiments to:

  • Measure GLI1 and PTCH1 expression by qPCR after SAG treatment

  • Compare SHH pathway activation between wildtype and FAM149B1-deficient cells

  • Assess ciliary localization of SHH pathway components using co-immunostaining

• Protein interaction studies:

  • Use co-immunoprecipitation with FAM149B1 antibodies to confirm interaction with TBC1D32

  • Perform proximity ligation assays to visualize interactions in situ

  • Compare protein interactomes between wildtype and disease-variant FAM149B1

What are the technical challenges in using FAM149B1 antibodies for distinguishing between different mutant variants?

Researchers face several technical challenges when using antibodies to distinguish FAM149B1 variants:

• Epitope accessibility issues: The reported disease variants (p.Gln118Hisfs20 and p.Gln147) create truncated proteins. Antibodies targeting regions beyond these truncation points will not detect the mutant proteins .

• Cross-reactivity with paralogs: FAM149A is an important paralog of FAM149B1 . Verify antibody specificity against this paralog, especially in tissues where both may be expressed.

• Detection of nonsense-mediated decay (NMD) effects: Many disease-causing variants may trigger NMD, resulting in absence of protein. In such cases, a lack of staining could indicate either antibody failure or true protein absence.

• Solution approach:

  • Use N-terminal-directed antibodies to detect truncated variants

  • Combine with RNA analysis to determine if NMD is occurring

  • Consider creating custom antibodies against specific mutant epitopes for variant-specific detection

How can FAM149B1 antibodies be applied in studies of ciliary trafficking and IFT dynamics?

To investigate ciliary trafficking using FAM149B1 antibodies:

• Live-cell imaging approach:

  • Create FAM149B1-fluorescent protein fusions for live tracking

  • Use anti-FAM149B1 antibodies to validate that fusion proteins localize correctly

  • Combine with IFT markers to assess co-transport

• IFT dynamics analysis:

  • Mutant FAM149B1 fibroblasts show abnormal accumulation of IFT88 and IFT46 (IFT-B complex members) at distal ciliary tips

  • Use immunostaining to quantify IFT-A (IFT122) vs IFT-B (IFT88, IFT46) distribution along the cilium

  • Assess retrograde transport defects through pulse-chase experiments

• Experimental protocol for IFT analysis:

  • Fix cells at different time points after serum starvation

  • Co-stain for FAM149B1, acetylated tubulin (ciliary axoneme), and IFT proteins

  • Quantify fluorescence intensity along the ciliary length using line scan analysis

  • Compare ratio of tip:shaft IFT protein intensity between wildtype and mutant cells

What approaches can resolve contradictory findings when using different FAM149B1 antibodies?

When faced with contradictory results from different FAM149B1 antibodies:

• Epitope mapping validation:

  • Determine precise epitope recognition using peptide arrays

  • Test antibodies against recombinant FAM149B1 fragments

  • Verify specificity using knockout/knockdown controls

• Methodological troubleshooting matrix:

• Integrated validation approach:

  • Combine antibody-based detection with genetic tagging methods

  • Use CRISPR/Cas9 epitope tagging to validate antibody binding sites

  • Apply multiple antibodies in the same experiment when possible

How can FAM149B1 antibodies contribute to phenotype-genotype correlation studies in ciliopathies?

FAM149B1 antibodies can significantly enhance phenotype-genotype studies through:

• Tissue-specific expression profiling:

  • Map FAM149B1 expression across affected tissues in ciliopathy patients

  • Compare expression patterns between different mutation types

  • Correlate protein expression levels with clinical severity

• Functional assessment of mutant proteins:

  • Use N-terminal antibodies to detect truncated proteins from frameshift mutations

  • Assess cellular localization of mutant proteins

  • Determine if mutants affect localization of interacting partners like TBC1D32

• Research protocol for patient fibroblasts:

  • Collect skin fibroblasts from patients with different FAM149B1 variants

  • Induce ciliogenesis through serum starvation

  • Analyze ciliary morphology, length, and IFT distribution

  • Correlate cellular phenotypes with clinical manifestations

  • Assess SHH pathway functionality using GLI1/PTCH1 expression

• Developmental timing considerations:

  • Mouse embryonic expression data shows strong expression in neuroepithelium at E8-10

  • Consider developmental timing when interpreting ciliopathy phenotypes

What methodologies can detect FAM149B1 abnormalities in clinical samples from ciliopathy patients?

For clinical research applications, consider these methodological approaches:

• Immunohistochemistry protocol for diagnostic tissues:

  • HIER pH 6 retrieval for paraffin-embedded tissues

  • Antibody dilutions of 1:200-1:500

  • Focus on tissues with known FAM149B1 expression (brain, retina, kidney)

• Fibroblast assay development:

  • Patient-derived fibroblasts can be analyzed for:

    • Ciliary length (increased in FAM149B1 mutants)

    • Ciliary morphology (bulbous tips)

    • IFT protein distribution

    • SHH pathway activation

• Biomarker correlation analysis:

  • Combine FAM149B1 antibody staining with disease markers

  • Assess correlation with clinical severity measures

  • Develop scoring systems for standardized assessment

• Tissue panel recommendations based on known FAM149B1-related phenotypes:

  • Neural tissue (for Joubert syndrome features)

  • Retina (for ocular manifestations including Duane syndrome)

  • Skeletal elements (for polydactyly and skeletal dysplasia)

  • Olfactory bulb (commonly aplastic in FAM149B1 ciliopathy)