FAM3A Antibody

What are the primary applications of FAM3A antibodies in current research?

FAM3A antibodies are primarily used in immunohistochemistry (IHC), immunofluorescence (ICC-IF), and Western blot analyses to study FAM3A localization and expression. These techniques have been crucial in establishing FAM3A's role in mitochondrial function, ATP synthesis regulation, and cell survival pathways. When selecting an antibody, researchers should verify that it has been validated for their specific application.

Optimal applications based on published research include:

• Western blot: For detecting FAM3A protein levels in tissue/cell lysates (0.2-1 μg/mL)

• Immunofluorescence: For subcellular localization studies (0.25-2 μg/mL)

• Immunohistochemistry: For tissue distribution analysis

Research has confirmed FAM3A localization predominantly in mitochondria, with expression in multiple tissues including kidney tubules, liver, pancreatic islets, and vascular tissues .

How should researchers validate FAM3A antibody specificity for their experimental model?

Methodological validation of FAM3A antibodies should include:

• Positive controls: Use tissues/cells known to express FAM3A (kidney tubules, pancreatic islets)

• Negative controls: Use FAM3A knockout tissues/cells or siRNA-treated samples

• Peptide competition assays: Pre-incubate antibody with immunizing peptide

• Multiple antibody comparison: Use antibodies targeting different epitopes of FAM3A

• Western blot analysis: Confirm single band at expected molecular weight (approximately 25-30 kDa)

Researchers should note that FAM3A knockout validation has been successfully performed using Ggt1-cre mice crossed with FAM3A-floxed mice to generate tubule-specific FAM3A knockout models . When validating antibodies for human samples, comparison with mRNA expression data from matching tissues can provide additional confirmation of specificity.

What are the optimal sample preparation methods for FAM3A antibody applications?

For Western blot analysis:

• Tissue homogenization should be performed in RIPA buffer supplemented with protease inhibitors

• For mitochondrial enrichment: Isolate mitochondria using differential centrifugation

• Protein loading: 20-50 μg of total protein per lane is typically sufficient

• Sample heating: 95°C for 5 minutes in reducing sample buffer

• Blocking: 5% non-fat dry milk in TBST (1 hour at room temperature)

For immunohistochemistry/immunofluorescence:

• Fixation: 4% paraformaldehyde (10-15 minutes)

• Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking: 5-10% normal serum (species different from primary antibody) with 0.1-0.3% Triton X-100

• Antibody dilution: 0.25-2 μg/mL (optimize for each specific antibody)

• Incubation: Overnight at 4°C

When studying mitochondrial localization, co-staining with mitochondrial markers such as TOMM20 is recommended, as demonstrated in research showing FAM3A downregulation in tubules with decreased TOMM20 .

How can researchers optimize FAM3A antibody signal in tissues with low expression levels?

For tissues with low FAM3A expression:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) system

  • Polymer-based detection systems

  • Avidin-biotin complex (ABC) method

• Microscopy optimization:

  • Use confocal microscopy with increased laser power and detector gain

  • Z-stack imaging with maximum intensity projection

  • Deconvolution to improve signal-to-noise ratio

• Sample preparation considerations:

  • Freshly collected samples yield better results than archival material

  • Shorter fixation times (4-6 hours) may preserve antigenicity

  • More aggressive antigen retrieval (extended time or higher temperature)

• Control experiments:

  • Include positive control tissues with known high FAM3A expression

  • Use FAM3A overexpression systems as technical positive controls

Recent studies demonstrated successful detection of low FAM3A levels in ischemic tissues using polymer-based detection systems with extended chromogen development times .

How should researchers design experiments to investigate the relationship between FAM3A and mitochondrial function?

When studying FAM3A's role in mitochondrial function, consider this experimental framework:

• FAM3A manipulation strategies:

  • Genetic approaches: CRISPR/Cas9 knockout, conditional knockout models (using Ggt1-cre as demonstrated) , or siRNA knockdown

  • Overexpression approaches: Viral vectors (AAV, lentivirus) or plasmid transfection

  • Pharmacological interventions: Test agents that affect FAM3A-related pathways (PI3K/AKT/NRF2)

• Mitochondrial function assessments:

  • ATP production measurement (luminescence-based assays)

  • Mitochondrial membrane potential (TMRM, JC-1 dyes)

  • Mitochondrial ROS production (MitoSOX Red)

  • Oxygen consumption rate (Seahorse XF analyzer)

  • Mitochondrial morphology (electron microscopy, confocal imaging with MitoTracker)

• Signaling pathway analysis:

  • Western blot for key proteins: p-PI3K, p-AKT, p-NRF2

  • Co-immunoprecipitation to detect protein-protein interactions

  • Subcellular fractionation to assess nuclear translocation of NRF2

• Functional readouts:

  • Cell viability assays

  • Pyroptosis markers (NLRP3, caspase-1, GSDMD-N)

  • Inflammatory cytokine production (IL-1β, IL-18)

Published research has demonstrated that FAM3A promotes PI3K/AKT/NRF2 signaling to block mitochondrial ROS accumulation and reduce NLRP3 inflammasome activation .

What methodological approaches are recommended for studying FAM3A in acute kidney injury models?

For researching FAM3A in acute kidney injury:

• Animal models selection:

  • Ischemia-reperfusion injury (IRI): Bilateral renal pedicle clamping for 30-45 minutes

  • Nephrotoxic models: Cisplatin (20 mg/kg), gentamicin, or folic acid administration

  • Contrast-induced nephropathy: Iodinated contrast agent injection

• FAM3A genetic manipulation in vivo:

  • Tubule-specific conditional knockout: Ggt1-cre × FAM3A-floxed mice

  • Overexpression: Hydrodynamic-based gene delivery of FAM3A expression plasmid (2 μg/g in 2 ml volume via tail vein)

  • Viral delivery: AAV vectors for targeted expression

• Assessment timeline:

  • Early phase: 6 hours post-injury

  • Peak injury: 24-48 hours

  • Recovery phase: 7 days

• Key measurements:

  • Renal function: Serum creatinine, BUN

  • Tubular injury markers: KIM-1, NGAL

  • Pyroptosis markers: NLRP3, caspase-1, GSDMD-N

  • Oxidative stress: mt-ROS, MDA levels

  • Inflammatory infiltration: Macrophage (F4/80+) and neutrophil (Ly6G+) quantification

  • ATP content in renal tissue

• Urinary biomarker analysis:

  • FAM3A quantification by ELISA or Western blot

  • Correlation with established AKI markers (IL-18, NGAL, β2-MG)

Research has shown that urinary FAM3A is significantly increased in AKI patients and correlates positively with other tubular injury markers, suggesting potential as a biomarker .

How should researchers interpret contradictory FAM3A expression patterns across different tissue types?

When facing contradictory FAM3A expression patterns:

• Tissue-specific regulatory mechanisms:

  • Analyze tissue-specific transcription factors that might regulate FAM3A

  • Examine epigenetic modifications in FAM3A promoter region across tissues

  • Consider alternative splicing variations using RT-PCR with isoform-specific primers

• Methodological considerations:

  • Antibody cross-reactivity: Validate with multiple antibodies targeting different epitopes

  • RNA-protein correlation: Compare protein levels with mRNA expression (RT-qPCR)

  • Single-cell analysis: Use single-cell RNA-seq or single-cell Western techniques to resolve cellular heterogeneity

• Analytical approach:

  • Perform quantitative image analysis with standardized protocols

  • Use normalization to housekeeping genes/proteins appropriate for each tissue

  • Apply statistical methods that account for biological variability

  • Consider meta-analysis of multiple datasets

• Contextual factors:

  • Disease state influences: Different pathological conditions may alter expression patterns

  • Developmental timing: Expression can vary throughout developmental stages

  • Environmental factors: Hypoxia, inflammation, and nutrient status affect FAM3A levels

Research has demonstrated that FAM3A expression varies across tubular segments and is differentially regulated under ischemic conditions, with proximal tubules showing more pronounced downregulation compared to distal tubules .

What are the key considerations when analyzing FAM3A knockout phenotypes in different disease models?

When analyzing FAM3A knockout phenotypes:

• Model-specific considerations:

  • Global vs. conditional knockout: Different phenotypic manifestations

  • Acute vs. chronic models: Temporal dynamics of FAM3A function

  • Strain background effects: C57BL/6N may show different phenotypes than other strains

  • Sex-specific differences: Analyze male and female animals separately

• Compensatory mechanisms:

  • Assess other FAM3 family members (FAM3B, FAM3C, FAM3D) for potential upregulation

  • Evaluate alternative pathways that might compensate for FAM3A loss

  • Time-course analysis to identify early vs. late compensation

• Molecular phenotyping:

  • Comprehensive pathway analysis: RNA-seq, proteomics, metabolomics

  • Mitochondrial function parameters: ATP production, oxidative phosphorylation

  • Stress response markers: Oxidative stress, ER stress, inflammatory markers

• Data integration framework:

  • Create multi-parameter scoring systems to quantify phenotype severity

  • Use principal component analysis to identify key variables driving phenotypic differences

  • Develop predictive models to understand FAM3A's role in disease progression

Studies have shown that FAM3A knockout exacerbates liver damage after ischemia-reperfusion with increased oxidative stress, decreased ATP content, and reduced Akt activity , while in kidney injury models, FAM3A loss accelerates tubular pyroptosis through decreased PI3K/AKT/NRF2 signaling .

What approaches can resolve discrepancies in FAM3A localization between different detection methods?

When facing discrepancies in FAM3A localization:

• Subcellular fractionation approach:

  • Perform differential centrifugation to isolate mitochondria, cytosol, nucleus, and other organelles

  • Analyze FAM3A distribution by Western blot with compartment-specific markers:

    • Mitochondria: TOMM20, COXIV

    • Cytosol: β-actin, GAPDH

    • Nucleus: Histone H3, Lamin B

    • ER: Calnexin

  • Quantify relative distribution ratios across compartments

• Microscopy method optimization:

  • Super-resolution techniques (STED, STORM) for improved spatial resolution

  • Live-cell imaging with FAM3A-fluorescent protein fusions

  • Co-localization analysis with multiple organelle markers

  • Electron microscopy with immunogold labeling for definitive localization

• Fixation and permeabilization variables:

  • Test multiple fixation methods (PFA, methanol, acetone)

  • Optimize permeabilization conditions for different compartments

  • Use epitope retrieval techniques appropriate for the subcellular compartment

• Antibody validation for each method:

  • Confirm specificity in each application using knockout controls

  • Use antibodies targeting different epitopes to confirm findings

  • Consider potential masking of epitopes in specific cellular contexts

Research confirms FAM3A localization primarily in mitochondria using multiple complementary approaches, including mitochondrial isolation and co-localization with TOMM20 .

How can researchers accurately quantify changes in FAM3A expression in response to stress or disease conditions?

For accurate quantification of FAM3A expression changes:

• Multi-level quantification strategy:

  • Protein level: Western blot with densitometry, ELISA, mass spectrometry

  • mRNA level: RT-qPCR, RNA-seq, nCounter analysis

  • Single-cell level: scRNA-seq, CyTOF, single-cell Western blot

  • Tissue level: Digital image analysis of immunostaining

• Normalization considerations:

  • For Western blot: Total protein normalization (REVERT stain) preferable to single housekeeping proteins

  • For RT-qPCR: Multiple reference genes (GAPDH, β-actin, 18S rRNA)

  • For immunostaining: Area-based normalization, internal control regions

• Temporal dynamics assessment:

  • Time-course analysis: Multiple timepoints (6h, 24h, 48h, 7d post-stimulus)

  • Pulse-chase experiments to determine protein half-life

  • Real-time monitoring using reporter systems

• Statistical analysis recommendations:

  • Use fold-change relative to appropriate controls

  • Apply ANOVA with post-hoc tests for multiple timepoints

  • Consider non-parametric methods for smaller sample sizes

  • Power analysis to determine adequate sample size

Studies demonstrate that FAM3A decreases at early stages (6h) in AKI mice and continues declining following disease progression (24h, 48h, 7d), highlighting the importance of temporal analysis .

What are the optimal experimental designs for investigating FAM3A's role in the PI3K/AKT/NRF2 signaling pathway?

For investigating FAM3A in PI3K/AKT/NRF2 signaling:

• Genetic manipulation matrix:

  • FAM3A manipulation: Knockout, knockdown, overexpression

  • Pathway component manipulation: PI3K inhibition/activation, AKT inhibition/activation, NRF2 knockout/activation

  • Combined approaches: FAM3A overexpression + PI3K inhibition, FAM3A knockout + NRF2 activation

• Biochemical analysis framework:

  • Phosphorylation status: p-PI3K, p-AKT, p-NRF2 by Western blot

  • Nuclear translocation: Nuclear/cytoplasmic fractionation for NRF2

  • Transcriptional activity: Luciferase reporter assays for NRF2-regulated genes

  • Protein-protein interactions: Co-immunoprecipitation, proximity ligation assay

• Functional readouts:

  • Antioxidant response: NRF2 target gene expression (NQO1, HO-1, GCLC)

  • ROS production: mt-ROS measurement using MitoSOX

  • Cell survival: Pyroptosis markers, apoptosis assessment

  • ATP production: Luminescence-based assays

• Pharmacological interventions:

  • NRF2 activators: Sulforaphane, Olipraz (NRF2 activator)

  • PI3K/AKT modulators: LY294002 (PI3K inhibitor), SC79 (AKT activator)

  • Purinergic receptor ligands: ADP or ATP supplementation

Published research demonstrated that NRF2 activator (Olipraz) alleviated pro-pyroptotic effects of FAM3A depletion, whereas NRF2 deletion blocked the anti-pyroptotic function of FAM3A, confirming the pathway relationship .

What considerations are important when designing FAM3A antibody-based detection methods for clinical biomarker applications?

For clinical biomarker applications of FAM3A:

• Sample type considerations:

  • Urine: Non-invasive but subject to variability; normalize to creatinine

  • Serum/plasma: Less direct for kidney injury but more stable

  • Tissue: Most direct but requires invasive sampling

• Detection method optimization:

  • ELISA development: Sandwich ELISA using antibodies targeting different epitopes

  • Point-of-care testing: Lateral flow immunoassay development

  • Multiplexed detection: Combined FAM3A with established markers (NGAL, KIM-1)

• Pre-analytical variables:

  • Sample collection timing: FAM3A peaks early in injury (6-24h)

  • Sample processing: Standardized centrifugation protocols

  • Storage conditions: -80°C with protease inhibitors to prevent degradation

• Clinical validation approach:

  • Correlation with established markers: IL-18, NGAL, β2-MG

  • Prognostic value assessment: ROC curve analysis, sensitivity/specificity determination

  • Longitudinal sampling: Serial measurements to establish temporal dynamics

• Analytical performance requirements:

  • Limit of detection: <5 ng/mL typically needed

  • Dynamic range: 10-1000 ng/mL to capture physiological and pathological levels

  • Precision: CV <10% for clinical applications

  • Specificity: No cross-reactivity with other FAM3 family members

Research has demonstrated that urinary FAM3A is significantly increased in AKI patients and positively correlates with IL-18, NGAL, and β2-MG levels, supporting its potential as a biomarker .

How should researchers design experiments to explore the therapeutic potential of FAM3A modulation in disease models?

For exploring FAM3A's therapeutic potential:

• Delivery system development:

  • Viral vectors: AAV serotypes with tissue tropism (AAV9 for heart, AAV8 for liver, AAV2 for kidney)

  • Non-viral approaches: Hydrodynamic-based gene delivery (2 μg/g in 2 ml volume)

  • Protein-based: Recombinant FAM3A with cell-penetrating peptides

  • Small molecule screening: Compounds that upregulate endogenous FAM3A

• Treatment regimen optimization:

  • Timing: Preventive vs. therapeutic administration

  • Dosing: Dose-response studies to determine optimal expression levels

  • Duration: Single vs. repeated administration

• Multi-disease model testing:

  • Kidney: Ischemia-reperfusion injury, nephrotoxic injury, diabetic nephropathy

  • Liver: Hepatic IRI, fatty liver disease, fibrosis models

  • Cardiovascular: Abdominal aortic aneurysm models, myocardial infarction

  • Neurological: Glutamate toxicity models, stroke models

  • Metabolic: Diabetes models, insulin resistance

• Outcome assessment framework:

  • Molecular endpoints: PI3K/AKT/NRF2 pathway activation, ATP levels

  • Cellular endpoints: Pyroptosis markers, mitochondrial function

  • Tissue endpoints: Histopathological scoring, inflammatory infiltration

  • Functional endpoints: Organ-specific function tests (GFR, insulin secretion)

Research has demonstrated successful FAM3A overexpression using hydrodynamic-based gene delivery, which alleviated kidney injury, inhibited pyroptosis, and reduced inflammatory cell infiltration in ischemia-reperfusion injury models .

What are the methodological approaches to resolve contradictions in FAM3A function across different cell types and disease models?

To resolve contradictions in FAM3A function:

• Systematic experimental framework:

  • Cell type matrix studies: Use identical experimental conditions across multiple cell types

  • Cross-disease model comparison: Apply consistent FAM3A manipulation across disease models

  • Age and sex considerations: Compare function in male vs. female, young vs. aged models

• Molecular mechanism dissection:

  • Cell-specific interaction partners: Identify unique FAM3A-interacting proteins by IP-MS

  • Post-translational modifications: Phosphorylation, ubiquitination, acetylation status

  • Isoform expression: Characterize cell-specific expression of FAM3A splice variants

  • Subcellular distribution: Compare mitochondrial vs. extracellular FAM3A functions

• Integrative multi-omics approach:

  • Transcriptomics: RNA-seq to identify cell-specific transcriptional responses

  • Proteomics: Phosphoproteomics to map signaling network differences

  • Metabolomics: Identify differential metabolic impacts of FAM3A

  • Network analysis: Construct cell-specific FAM3A-centered interaction networks

• Translational investigations:

  • Human vs. mouse differences: Compare FAM3A function across species

  • Healthy vs. disease state: Analyze functional differences in pathological contexts

  • Age-dependent alterations: Compare FAM3A function throughout lifespan