FAM195A Antibody

What is FAM195A and why is it important for metabolic research?

FAM195A is a recently discovered RNA binding protein that plays a crucial role in cold-induced thermogenesis. Research shows that FAM195A is highly expressed in brown adipose tissue (BAT) and striated muscles, with significantly lower expression in white adipose tissues. Its importance stems from its regulatory function in branched-chain amino acid (BCAA) metabolism and fatty acid oxidation, which are essential for BAT activation and thermogenesis .

Genetically engineered mice lacking FAM195A display severe cold intolerance and abnormalities in BCAA metabolism, demonstrating FAM195A's critical role in thermoregulation. The protein establishes a previously unrecognized link between thermoregulation, metabolism, and RNA processes . This makes FAM195A a potential therapeutic target for metabolic disorders, particularly obesity and related conditions where enhancing energy expenditure through BAT activation represents a promising strategy.

What tissue types should be prioritized when validating FAM195A antibodies?

When validating FAM195A antibodies, researchers should prioritize brown adipose tissue (BAT), skeletal muscle, and cardiac muscle as primary validation tissues due to their high expression levels of FAM195A . The protein shows strong expression in interscapular BAT and all muscle groups, with minimal detection in white adipose tissues such as inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) .

While some FAM195A protein has been detected in liver and adrenal gland samples, the signal in adrenal tissue may be attributed to BAT surrounding the adrenal structure, as confirmed by UCP1 expression in the same samples . For negative controls in antibody validation, white adipose tissues serve as excellent low-expression tissues. Additionally, samples from FAM195A knockout models, if available, provide definitive negative controls for antibody specificity assessment.

What are the recommended applications for FAM195A antibody detection?

Based on published research, FAM195A antibodies have been successfully employed in several experimental applications:

• Western blot analysis: Commercial antibodies against full-length FAM195A have demonstrated efficacy in detecting the protein in tissue lysates, with clear differential expression across tissue types .

• Immunocytochemistry/Immunofluorescence: FAM195A antibodies work effectively for cellular localization studies, revealing both diffuse cytosolic and nuclear patterns in differentiated adipocytes, with distinct puncta formation in cytosol upon stress granule induction .

• Subcellular fractionation studies: FAM195A antibodies can detect the protein in both cytosolic and nuclear fractions, with predominant expression in the cytosolic compartment .

• RNA-protein interaction studies: When combined with techniques like UV cross-linking and immunoprecipitation, FAM195A antibodies can help investigate the RNA binding properties of the protein .

For optimal results, researchers should validate each application independently, as antibody performance may vary between techniques.

How can I determine if my FAM195A antibody is specifically detecting the target protein?

To confirm FAM195A antibody specificity, implement the following validation approach:

• Tissue expression pattern verification: Confirm that the antibody detects higher signal in tissues known to express FAM195A abundantly (BAT, skeletal muscle) compared to tissues with low expression (white adipose tissue) .

• Molecular weight confirmation: Verify that the detected band on Western blots matches the expected molecular weight of FAM195A.

• Genetic models: Where available, compare antibody detection between wild-type tissues and those from FAM195A knockout animals, which should show absence of signal in knockout samples .

• RNA interference validation: In cell culture models, compare antibody detection between control cells and those where FAM195A has been knocked down using shRNA or siRNA approaches. Research has demonstrated that shRNA-mediated knockdown (particularly shRNA-2 and shRNA-4) effectively reduces FAM195A at both transcript (50-70% reduction) and protein levels .

• Recombinant protein controls: If using tagged versions of FAM195A (such as FLAG-tagged or TY1-tagged FAM195A), confirm that the antibody detects both endogenous and tagged versions at their respective molecular weights .

What controls should be included when using FAM195A antibodies for immunolocalization?

When performing immunolocalization studies with FAM195A antibodies, include these essential controls:

• Positive tissue controls: Include BAT and skeletal muscle sections, which express high levels of FAM195A .

• Negative tissue controls: Include white adipose tissue samples, where FAM195A expression is minimal .

• Primary antibody omission: Process some sections without primary antibody to identify potential non-specific binding of secondary antibodies.

• Blocking peptide competition: Pre-incubate the antibody with its immunizing peptide to demonstrate signal specificity.

• Subcellular localization verification: Confirm that the observed pattern matches the expected diffuse cytosolic and nuclear distribution described in literature . Under stress conditions (e.g., sodium arsenite treatment), verify the formation of distinct puncta corresponding to stress granules .

• Co-localization studies: For advanced validation, perform double immunostaining with markers of known FAM195A-associated structures, such as DDX6 in RNA stress granules .

How does FAM195A protein expression change during adipocyte differentiation and cold exposure?

FAM195A exhibits dynamic expression patterns during adipogenesis and in response to thermogenic stimuli:

When designing experiments to study FAM195A regulation, researchers should consider time-course analyses that capture these dynamic changes. Antibody-based detection methods should include sampling at multiple timepoints following differentiation or cold exposure. Additionally, FAM195A expression corresponds with activation of the brown/beige fat program, as its promoter is bound by transcriptional regulators Ebf2 and Pparg, which maintain brown and/or beige fat cell identity .

For optimal detection of these changes, use antibodies validated for both Western blot and immunofluorescence applications to capture both quantitative and spatial expression patterns.

What are the key technical considerations when optimizing Western blot protocols for FAM195A detection?

Successful Western blot detection of FAM195A requires careful optimization of several parameters:

• Sample preparation: For BAT samples, thorough homogenization in the presence of protease inhibitors is critical due to the high lipid content. Centrifugation steps should be optimized to remove lipid contaminants without losing protein yield.

• Protein amount: Load 30-50μg of total protein per lane for tissues with high FAM195A expression (BAT, muscle) and consider increasing to 50-75μg for tissues with lower expression .

• Subcellular fractionation: When studying FAM195A's distribution between cytosolic and nuclear compartments, sequential extraction protocols are recommended, with verification of fraction purity using established markers (e.g., GAPDH for cytosol, Lamin B for nucleus) .

• Antibody dilution: Based on published protocols, commercial FAM195A antibodies are typically effective at 1:1000 dilution for Western blot applications, but optimal concentration should be determined empirically for each antibody.

• Detection system: Enhanced chemiluminescence (ECL) systems are suitable for FAM195A detection, but for quantitative comparisons across multiple tissues with varying expression levels, consider digital fluorescence-based detection platforms for better linear range.

• Normalization controls: When comparing FAM195A levels between tissues or conditions, select loading controls appropriate for each tissue type, as traditional housekeeping proteins may vary between adipose and muscle tissues.

How can I investigate FAM195A's protein-protein interactions and RNA binding properties?

To study FAM195A's interactions and RNA binding functions, consider these methodological approaches:

• Proximity labeling assays: The FAM195A-miniTurbo fusion protein approach has been successfully employed to identify proteins in close proximity to FAM195A . This technique involves:

  • Generating a construct with biotin ligase (miniTurbo) fused to FAM195A

  • Expressing the fusion protein in relevant cell types (e.g., SVF-BAT cells)

  • Pulsing with exogenous biotin

  • Purifying biotinylated proteins via streptavidin

  • Analyzing by mass spectrometry

• RNA binding assays: To confirm FAM195A's RNA binding properties, implement polynucleotide kinase-mediated 32P labeling of RNA after immunoprecipitation . This approach requires:

  • Expression of tagged FAM195A (e.g., TY1-FAM195A)

  • UV cross-linking to stabilize protein-RNA interactions

  • Immunoprecipitation of the protein-RNA complexes

  • RNA labeling and SDS-PAGE analysis

• RNA stress granule association: To study FAM195A localization to stress granules:

  • Treat cells with translation inhibitors (e.g., sodium arsenite)

  • Perform immunofluorescence staining for FAM195A and stress granule markers

  • Analyze co-localization using confocal microscopy

When designing these experiments, include appropriate controls such as non-cross-linked samples, RNase-treated samples, and known RNA binding proteins (e.g., hnRNP/C) as technical controls .

What are the implications of FAM195A's function for interpreting antibody-based studies in metabolic research?

Understanding FAM195A's function has important implications for experimental design and interpretation in metabolic research:

• Tissue-specific expression considerations: When studying FAM195A in mixed tissues (e.g., adrenal gland with surrounding BAT), carefully dissect and verify tissue identity, as FAM195A signal may originate from adipose contamination rather than the target organ .

• Metabolic pathway integration: FAM195A regulates both BCAA metabolism and fatty acid oxidation pathways, particularly affecting enzymes like IVD (Isovaleryl-CoA dehydrogenase), BCAT2 (Branched-chain amino acid transaminase 2), and CPT2 (Carnitine palmitoyltransferase 2) . When studying these metabolic pathways, consider FAM195A as a potential regulator affecting results.

• Cold exposure protocols: In studies involving cold exposure, FAM195A expression changes may represent an adaptation mechanism. Monitor both FAM195A levels and its downstream targets to fully interpret thermogenic responses. Note that FAM195A knockout mice become severely hypothermic (<30°C) as early as 2 hours after acute cold exposure .

• RNA processing connections: FAM195A interacts with protein complexes involved in mRNA processing, suggesting that its effects on metabolism may be mediated through post-transcriptional regulation . When investigating gene expression changes in relevant pathways, consider analyzing both transcript and protein levels.

• Functional readouts: Beyond simple protein detection, consider measuring functional outcomes associated with FAM195A activity, such as oxygen consumption rates, BCAA metabolism enzyme activities, and thermogenic capacity.

How can I overcome challenges in detecting FAM195A in co-immunoprecipitation and chromatin immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) with FAM195A antibodies present unique challenges:

• Cross-linking optimization for RNA binding proteins:

  • For Co-IP of FAM195A-protein complexes, mild formaldehyde cross-linking (0.1-0.2%) may preserve interactions

  • For RNA-associated complexes, UV cross-linking at 254nm is recommended as demonstrated in published protocols

  • Test multiple cross-linking conditions, as excessive cross-linking may mask antibody epitopes

• Buffer composition considerations:

  • Include RNase inhibitors when studying RNA-protein complexes

  • Test various detergent concentrations (0.1-1% NP-40 or Triton X-100) to balance complex preservation and background reduction

  • For nuclear complexes, include appropriate nuclear extraction buffers with salt concentrations optimized for nuclear protein-protein interactions

• Antibody orientation strategies:

  • Compare results using antibodies targeting different epitopes of FAM195A

  • Consider tagged versions of FAM195A (FLAG, TY1) for pull-down experiments when antibody efficiency is limiting

  • For the reverse approach, identify specific interacting partners (e.g., from the 81 proteins identified in proximity labeling studies ) and use antibodies against these partners for Co-IP

• Verification approaches:

  • Perform reciprocal Co-IPs when possible

  • Include stringent controls (IgG, non-interacting proteins)

  • Validate interactions using orthogonal methods such as proximity ligation assay or FRET in intact cells

• Advanced considerations for ChIP experiments:

  • Since FAM195A is an RNA binding protein rather than a direct DNA binding protein, consider RNA-ChIP or ChIRP (Chromatin Isolation by RNA Purification) to identify RNA-mediated chromatin associations

  • For conventional ChIP, focus on protein complexes known to interact with FAM195A that have DNA binding capacity

How can FAM195A antibodies be applied in obesity and metabolic disorder research?

FAM195A antibodies offer valuable tools for investigating the molecular mechanisms underlying obesity and metabolic disorders:

• BAT activation assessment: Use FAM195A antibodies to monitor brown adipose tissue activation status in experimental models of obesity and during therapeutic interventions. The "whitening" of BAT observed in FAM195A knockout mice suggests that FAM195A expression correlates with BAT thermogenic capacity .

• Metabolic pathway analysis: Employ FAM195A antibodies in multiplex immunoassays alongside markers of BCAA metabolism and fatty acid oxidation to comprehensively assess metabolic pathway dysregulation in disease states. This approach can reveal whether FAM195A downregulation contributes to metabolic dysfunction through specific enzymatic deficiencies.

• Therapeutic target evaluation: In drug discovery research, FAM195A antibodies can serve as pharmacodynamic markers to evaluate compounds designed to enhance BAT activity. Changes in FAM195A expression or localization may indicate successful target engagement of thermogenic pathways.

• Correlation studies with clinical parameters: In translational research involving human samples, quantify FAM195A levels in relation to clinical parameters such as BMI, insulin sensitivity, and cold tolerance to establish potential biomarker applications.

• Intervention response monitoring: Use FAM195A antibodies to track molecular responses to interventions known to activate BAT, such as cold exposure, β3-adrenergic agonists, or PPARγ activators like rosiglitazone .

What methodological approaches can resolve contradictory findings in FAM195A expression studies?

When faced with contradictory results regarding FAM195A expression or function, implement these methodological strategies:

• Antibody validation matrix:

• Environmental condition standardization: Control for factors known to influence FAM195A expression, including:

  • Housing temperature (standard 22°C vs. thermoneutral 30°C conditions)

  • Feeding status (fed vs. fasted)

  • Circadian timing of tissue collection

  • Acute vs. chronic cold exposure protocols

• Technical approach diversification:

  • Complement antibody-based detection with RNA-based methods (qRT-PCR, RNA-seq)

  • Use multiple antibodies targeting different epitopes

  • Employ orthogonal protein detection methods (mass spectrometry)

  • Include functional readouts of FAM195A activity (BCAA metabolism, thermogenic capacity)

• Comprehensive data reporting:

  • Document complete methodological details, including antibody source, catalog number, and dilution

  • Report both positive and negative findings

  • Include raw data and representative images

  • Provide detailed information about sample processing timelines

How can FAM195A antibodies be used to investigate the connection between thermogenesis and RNA processing?

FAM195A provides a unique opportunity to study the intersection of thermogenesis and RNA metabolism. Consider these advanced methodological approaches:

• RNA-protein complex visualization: Employ immunofluorescence with FAM195A antibodies combined with RNA FISH to visualize FAM195A interaction with specific target RNAs in intact cells under various thermogenic conditions.

• Stress granule dynamics analysis: Use FAM195A antibodies to track stress granule formation during cold adaptation:

  • Monitor FAM195A localization changes at different timepoints during cold exposure

  • Quantify co-localization with RNA stress granule markers like DDX6

  • Assess how these dynamics correlate with thermogenic gene expression

• Target mRNA stability assessment:

  • Perform FAM195A immunoprecipitation followed by RNA sequencing to identify bound transcripts

  • Focus analysis on transcripts encoding BCAA metabolism enzymes found to be downregulated in FAM195A knockout mice

  • Conduct mRNA stability assays in the presence or absence of FAM195A to determine its role in post-transcriptional regulation

• Subcellular fractionation studies:

  • Use FAM195A antibodies to track protein redistribution between cytosolic and nuclear compartments during thermogenic activation

  • Correlate these changes with alterations in RNA processing events

  • Compare patterns in BAT versus white adipose undergoing browning

• Translation regulation analysis:

  • Investigate whether FAM195A associates with translating ribosomes during cold adaptation

  • Examine polysome profiles in wild-type versus FAM195A knockout cells

  • Assess translation efficiency of BCAA metabolism enzyme mRNAs using ribosome profiling

How can new antibody technologies enhance FAM195A research beyond traditional applications?

Emerging antibody technologies offer exciting opportunities to advance FAM195A research:

• FAM195A-specific nanobodies:

  • Single-domain antibodies with superior tissue penetration for in vivo imaging

  • Smaller size allows access to restricted cellular compartments

  • Potential for intrabody applications to visualize FAM195A in living cells

• Proximity-dependent labeling approaches:

  • Antibody-enzyme fusions (APEX, BioID) to identify context-specific FAM195A interaction partners

  • Split-BioID systems to detect specific FAM195A interactions under different metabolic conditions

  • This builds upon the successful miniTurbo-FAM195A fusion approach already demonstrated

• Degrader technologies:

  • FAM195A-targeting PROTACs (Proteolysis Targeting Chimeras) for rapid protein depletion

  • Antibody-based degraders to achieve tissue-specific FAM195A knockdown

  • These approaches offer advantages over genetic knockout models by allowing temporal control

• Spatially-resolved single-cell antibody applications:

  • Imaging mass cytometry with FAM195A antibodies to map expression across tissue microenvironments

  • Single-cell Western blot technologies to quantify FAM195A heterogeneity within BAT

  • Spatial transcriptomics combined with FAM195A immunofluorescence to correlate protein levels with local gene expression patterns

• Conformational state-specific antibodies:

  • Development of antibodies that specifically recognize FAM195A in RNA-bound versus unbound states

  • Phospho-specific antibodies to detect post-translational modifications that may regulate FAM195A function

  • These tools could provide insights into the activation mechanisms of FAM195A during cold adaptation

What experimental designs can help resolve the temporal dynamics of FAM195A function during cold adaptation?

To fully characterize FAM195A's role in the adaptive response to cold exposure, consider these sophisticated experimental approaches:

• Time-resolved tissue sampling strategy:

• Multi-parameter imaging approach:

  • Serial tissue sections analyzed with FAM195A antibodies alongside markers for:

    • RNA stress granules (DDX6, G3BP1)

    • Mitochondrial activity (OXPHOS complexes)

    • BCAA metabolism enzymes (IVD, BCAT2)

    • BAT activation (UCP1, PGC1α)

  • Quantitative image analysis to establish temporal relationships between these parameters

• Inducible expression systems:

  • Design experiments with doxycycline-inducible FAM195A expression

  • Introduce FAM195A at different stages of cold adaptation in knockout models

  • Determine the critical windows during which FAM195A is essential for adaptive thermogenesis

• Metabolic flux analysis:

  • Combine FAM195A detection with metabolic tracing of BCAAs and fatty acids

  • Assess how FAM195A expression correlates with substrate utilization changes

  • Identify the rate-limiting steps in metabolic adaptation that depend on FAM195A

• Microfluidic approaches:

  • Culture adipocytes in temperature-controlled microfluidic devices

  • Apply antibody-based live cell imaging of FAM195A dynamics

  • Correlate cellular responses with real-time temperature changes

What are the most significant challenges in developing highly specific FAM195A antibodies for research applications?

Developing highly specific antibodies against FAM195A presents several technical challenges:

• Protein structure considerations:

  • FAM195A contains disordered domains which may adopt different conformations in various cellular contexts

  • These structural features can affect epitope accessibility and antibody recognition

  • Solution: Generate antibodies against multiple epitopes, including both disordered and structured regions

• Cross-reactivity with related proteins:

  • FAM195A may share sequence homology with other RNA binding proteins

  • This could lead to non-specific binding and false-positive results

  • Solution: Perform comprehensive cross-reactivity testing against related RNA binding proteins and validate in knockout models

• Post-translational modifications:

  • FAM195A function may be regulated by post-translational modifications

  • These modifications could mask epitopes or alter antibody binding

  • Solution: Characterize the post-translational modification landscape of FAM195A and develop modification-insensitive antibodies

• Antibody validation in complex tissues:

  • BAT contains high lipid content that can interfere with immunodetection

  • Signal specificity must be confirmed across multiple tissue types

  • Solution: Implement rigorous validation protocols including genetic controls and multiple detection methods

• Ensuring compatibility with multiple applications:

  • An ideal FAM195A antibody should work across applications (Western blot, immunohistochemistry, immunoprecipitation)

  • Different applications have distinct requirements for epitope accessibility

  • Solution: Screen antibody candidates across all intended applications early in development