FAM151B Antibody

Basic Research Questions

• What is FAM151B and why is it important for antibody-based research applications?

FAM151B is a mammalian homologue of the C. elegans menorin gene, which is involved in neuronal branching . It belongs to the PLC-like phosphodiesterase superfamily of enzymes that hydrolyze phosphodiester bonds of various substrates . FAM151B is critically important in retinal health, as knockout mice exhibit severe retinal degeneration and complete loss of photoreceptor function . Antibodies against FAM151B are essential for studying its expression patterns, localization, and functional role in normal and pathological conditions. The protein contains a DUF2181 domain with conserved active site residues that are crucial for its enzymatic function, making it an important target for structure-function studies .

• What tissue expression patterns should researchers expect when using FAM151B antibodies?

When using FAM151B antibodies, researchers should expect to detect low-level expression across multiple tissues, with notable expression in ocular tissues. According to BioGPS expression data, FAM151B is expressed at low levels in most tissues including the retinal pigmented epithelium (RPE), retina, iris, ciliary body, lens, and cornea . This contrasts with its paralogue FAM151A, which shows limited tissue distribution with high expression primarily in the intestine, kidney, and spleen . When designing immunohistochemistry or immunofluorescence experiments, researchers should consider these expression patterns and include appropriate positive and negative control tissues. Western blot analysis should be optimized for detection of potentially low abundance signals in most tissues except for ocular tissues where expression may be more prominent.

• How can researchers validate the specificity of commercial FAM151B antibodies?

Validation of FAM151B antibodies should follow a multi-step approach. First, perform Western blot analysis using tissue from wild-type animals alongside FAM151B knockout models to confirm antibody specificity . The availability of FAM151B knockout mice (such as the tm1b(EUCOMM)Hmgu line) provides an excellent negative control . Second, conduct immunohistochemistry or immunofluorescence on tissues with known expression patterns, comparing staining between wild-type and knockout samples. Third, validate through immunoprecipitation followed by mass spectrometry to confirm target identity. Fourth, test cross-reactivity with the paralogous FAM151A protein, particularly in tissues where both are expressed. Finally, perform peptide competition assays using the immunizing peptide to confirm binding specificity. Researchers should document lot-to-lot variation in commercial antibodies, as this can significantly impact experimental reproducibility.

• What are the recommended fixation and sample preparation protocols for FAM151B immunodetection?

For optimal FAM151B immunodetection, tissue fixation and processing must be carefully controlled. For immunohistochemistry of retinal tissues, 4% paraformaldehyde fixation for 24 hours followed by paraffin embedding is recommended, as demonstrated in studies of retinal degeneration in FAM151B knockout mice . For immunofluorescence, shorter fixation times (15-30 minutes) with 4% paraformaldehyde may better preserve epitope accessibility. When studying RPE tissues specifically, researchers should consider the protocol used for ZO-1 and RPE65 staining in FAM151B studies, where careful removal of the retina from the RPE allowed for whole-mount visualization of tight junctions . For Western blot applications, standard RIPA buffer extraction with protease inhibitors is suitable, but optimization may be required depending on the cellular localization and solubility of FAM151B in specific tissues. Flash-freezing tissues before protein extraction is recommended to minimize protein degradation. Epitope retrieval methods (heat-induced or enzymatic) should be optimized for fixed tissues to maximize antibody binding while minimizing background.

Advanced Research Questions

• How can FAM151B antibodies be used to investigate the temporal dynamics of photoreceptor degeneration?

FAM151B antibodies can be employed in a comprehensive temporal analysis of photoreceptor degeneration through carefully designed time-course experiments. Based on the FAM151B knockout mouse model, researchers should design sampling points at P11 (pre-eye opening), P15 (early degeneration with stress markers but intact cells), P21 (dramatic reduction in photoreceptors), and later time points to capture the complete degenerative process . Immunohistochemistry should be performed with FAM151B antibodies in conjunction with markers of retinal stress (GFAP), photoreceptor-specific proteins (rhodopsin, M/L-opsin), and cell death markers at each time point . Quantitative analysis should include measurement of outer nuclear layer thickness, outer segment length, and photoreceptor cell counts, correlated with FAM151B expression patterns . Combining immunofluorescence with ERG functional measurements at each time point allows correlation between protein expression/localization and functional deficits . This approach would reveal whether FAM151B loss precedes functional decline or whether compensatory mechanisms temporarily maintain function despite protein absence.

• What methodological considerations are important when using FAM151B antibodies to investigate its role in the PLC-like phosphodiesterase superfamily?

When investigating FAM151B's role in the PLC-like phosphodiesterase superfamily, researchers must consider several methodological factors. First, antibody selection should target conserved catalytic regions of the DUF2181 domain, which contains the evolutionarily conserved active site shared with other PLC-like phosphodiesterases . Second, implement phosphodiesterase activity assays in parallel with antibody-based detection to correlate protein presence with enzymatic function. Third, design co-immunoprecipitation experiments using FAM151B antibodies to identify interacting proteins within the phosphodiesterase pathway, particularly potential substrates. Fourth, develop comparative immunostaining protocols to visualize FAM151B alongside other family members, using confocal microscopy to assess co-localization. Fifth, consider using phospho-specific antibodies to determine if FAM151B undergoes post-translational modifications that regulate its enzymatic activity. Lastly, when analyzing data, researchers should be aware that FAM151B may hydrolyze various substrates like other family members, which range from GPI protein anchors to sphingolipids and lysolipids . This versatility requires careful experimental design to identify the physiologically relevant substrates in retinal tissue.

• How do researchers distinguish between FAM151A and FAM151B protein expression patterns and functions using antibody-based techniques?

Distinguishing between FAM151A and FAM151B requires careful antibody selection and experimental design due to potential structural similarities between these paralogues. First, researchers should select antibodies raised against non-conserved regions, particularly avoiding the duplicated DUF2181 domain found in FAM151A but not FAM151B . Validation using both FAM151A and FAM151B knockout tissues is essential to confirm specificity . When performing Western blot analysis, researchers should consider the expected molecular weight differences: FAM151A contains a duplicated domain structure that results in a larger protein compared to FAM151B . Immunohistochemistry experiments should include tissues with differential expression patterns (e.g., kidney for FAM151A and retina for FAM151B) as internal controls . For functional studies, researchers can take advantage of the distinct phenotypes of the knockout models; FAM151B knockout mice show severe retinal degeneration, while FAM151A knockout mice display no detectable retinal phenotype even at one year of age . Dual immunofluorescence labeling can help determine if the proteins are co-expressed in any tissues, which might indicate functional redundancy. Finally, careful quantification of immunoblot and immunostaining signals is necessary, as FAM151B is expressed at low levels in most tissues compared to the more tissue-restricted but higher expression of FAM151A in specific organs .

• What experimental approaches can integrate FAM151B antibody detection with the study of FAM151B-DT lncRNA in protein homeostasis pathways?

Integration of FAM151B protein and FAM151B-DT lncRNA studies requires sophisticated experimental design. First, perform dual-labeling experiments using FAM151B antibodies alongside RNA fluorescence in situ hybridization (FISH) for FAM151B-DT to determine spatial correlation between the protein and lncRNA . Second, implement RNA immunoprecipitation (RIP) assays using FAM151B antibodies to assess direct interactions between the protein and its namesake lncRNA . Third, design co-immunoprecipitation experiments targeting FAM151B, tau, α-synuclein, and HSC70, as these proteins have been shown to interact with FAM151B-DT . Fourth, establish a quantitative correlation between FAM151B protein levels and FAM151B-DT expression in both normal and pathological tissues using Western blot and qRT-PCR in parallel. Fifth, employ proximity ligation assays (PLA) to visualize and quantify interactions between FAM151B and components of the autophagy machinery that are regulated by FAM151B-DT . Finally, conduct rescue experiments in FAM151B-DT knockdown models using exogenous FAM151B protein to determine functional relationships. This multimodal approach would reveal whether FAM151B-DT regulates FAM151B protein function or whether they operate in parallel pathways affecting protein homeostasis.

• How can FAM151B antibodies be utilized in tau aggregation studies and neurodegenerative disease models?

FAM151B antibodies can be strategically employed in tau aggregation research through several approaches. First, utilize immunofluorescence co-labeling with FAM151B antibodies and phospho-tau markers in brain tissue from tauopathy patients (FTLD-tau, PSP, AD) where FAM151B-DT levels are reduced . Second, implement proximity ligation assays to investigate potential physical interactions between FAM151B protein and tau or α-synuclein, particularly in the context of FAM151B-DT manipulation . Third, perform sequential extraction protocols to separate soluble and insoluble tau fractions, followed by immunoblotting for both FAM151B and tau to determine correlation between FAM151B localization and tau aggregation states . Fourth, design time-course experiments in tau biosensor cell models with FAM151B-DT knockdown, using FAM151B antibodies to track protein expression changes alongside tau seeding measurements . Fifth, conduct immunoprecipitation with FAM151B antibodies followed by mass spectrometry to identify the complete interactome in normal versus tauopathy conditions, which may reveal additional protein homeostasis partners beyond HSC70 . Finally, perform quantitative analysis of autophagy markers in conjunction with FAM151B immunostaining in models where FAM151B-DT expression is manipulated, as this lncRNA has been shown to impact autophagy and protein degradation pathways vital for preventing protein aggregation .

• What advanced subcellular localization techniques using FAM151B antibodies can provide insights into its function in protein degradation pathways?

Advanced subcellular localization techniques using FAM151B antibodies can significantly enhance our understanding of its role in protein degradation. First, implement super-resolution microscopy (STORM, PALM, or SIM) with FAM151B antibodies and markers for autophagosomes (LC3), lysosomes (LAMP1), and proteasomes to achieve nanoscale resolution of FAM151B's association with degradation machinery . Second, use live-cell imaging with FAM151B antibody fragments or nanobodies to track dynamic associations with degradation compartments in real-time, particularly in response to stress conditions. Third, employ subcellular fractionation followed by Western blotting to quantitatively assess FAM151B distribution across cytosolic, membrane, nuclear, and organelle fractions, especially in relation to HSC70 and other chaperones . Fourth, conduct correlative light and electron microscopy (CLEM) using FAM151B antibodies to visualize its ultrastructural location relative to autophagic structures. Fifth, perform expansion microscopy to physically enlarge subcellular structures, allowing detailed mapping of FAM151B in relation to tau and α-synuclein aggregates . Sixth, implement multiplexed ion beam imaging (MIBI) or imaging mass cytometry to simultaneously visualize dozens of proteins in the degradation pathways alongside FAM151B. These techniques can reveal how FAM151B's subcellular localization changes during the progression of protein aggregation diseases and in response to FAM151B-DT modulation, providing crucial insights into its mechanistic role in protein homeostasis .