fam136a Antibody

What is FAM136A and where is it expressed in mammalian tissues?

FAM136A (Family with sequence similarity 136, member A) is a 16-kDa protein expressed widely across tissues. While initially considered to have unknown specific functions, recent research has characterized it as a mitochondrial protein involved in the electron transport chain of respiration . Expression analysis shows FAM136A is not limited to specific tissues but is present across various cell types with particular abundance in neurosensory epithelial cells . Similar to oxidative phosphorylation (OXPHOS) genes, FAM136A is expressed in all tissues examined as well as in most cancer cell lines, contradicting earlier assumptions that its expression might be restricted to the inner ear . Western blot detection has confirmed its presence in multiple human cell lines including U2OS, A549, HeLa, Jurkat, and MCF-7, as well as in mouse testis tissue .

What are the validated research applications for FAM136A antibodies?

FAM136A antibodies have been validated for multiple research applications with specific recommended dilution protocols:

Researchers should note that optimal dilutions may be sample-dependent, and preliminary titration experiments are recommended for each testing system to achieve optimal results .

How should researchers optimize immunohistochemical detection of FAM136A?

For optimal immunohistochemical detection of FAM136A, researchers should implement the following protocol refinements:

• Antigen retrieval: Use TE buffer at pH 9.0 as the primary method, with citrate buffer pH 6.0 as an alternative if needed .

• Primary antibody: A dilution range of 1:50-1:500 has been validated, but researchers should titrate to determine optimal concentration for their specific tissue samples .

• Detection system: The antigen-antibody complex can be visualized with 3,3'-diaminobenzidine (DAB) solution followed by hematoxylin counterstaining .

• Scoring methodology: Implement semi-quantitative scoring for extent of immunoreaction, such as: 0 (0% immunoreactive cells), 1 (<5%), 2 (5-50%), and 3 (>50%) .

• Controls: Include PBS as negative control alongside positive control tissues with known FAM136A expression .

When interpreting results, note that FAM136A is typically detected in the cytoplasm of carcinoma cells but may be focally and weakly immunolocalized in adjacent non-neoplastic epithelial cells or healthy tissue .

What experimental approaches can researchers use to study FAM136A function in mitochondrial homeostasis?

To investigate FAM136A's role in mitochondrial function, particularly in inter-membrane space protein homeostasis, researchers can implement several complementary approaches:

• Protein-protein interaction studies: Stable expression of tagged FAM136A (e.g., FAM136A-FLAG) followed by immunoprecipitation and mass spectrometry has successfully identified interactions with IMS-exposed proteins .

• CRISPR-Cas9 depletion experiments: Genetic knockout or knockdown of FAM136A followed by:

  • Proteomics analysis to assess global changes in protein abundance

  • Western blot validation of specific IMS proteins (HAX1, CLPB, ENDOG, NNT)

  • Assessment of respiratory chain complex proteins (e.g., NDUFB8, MT-CO1)

• Protein stability assays: Block cytosolic protein synthesis using cycloheximide and measure protein half-life of IMS proteins compared to matrix proteins (e.g., citrate synthase) in FAM136A-depleted vs. control cells .

• Functional respiratory measurements: Assess changes in cellular respiration following FAM136A depletion using respirometry techniques .

• In vivo validation: Analysis of tissues from Fam136a knockout mice, particularly focusing on age-related phenotypes that may mimic Ménière's disease .

How can researchers address discrepancies in FAM136A expression patterns between different experimental models?

When encountering contradictory FAM136A expression data between different experimental models, researchers should implement a systematic approach:

• Tissue-specific variability: Research has demonstrated that FAM136A depletion affects tissues differently. For example, OXPHOS protein abundance decreases were observed in the heart of Fam136a KO mice but not in brain or liver, suggesting tissue-specific sensitivity . Design experiments to include multiple tissue types or cell lines.

• Heterozygous vs. homozygous models: Consider that heterozygous FAM136A mutations (as seen in familial Ménière's disease) may present with tissue-restricted phenotypes, while homozygous inactivation (as in some research models) may produce multi-organ effects .

• Compensatory mechanisms: Look for upregulation of stress response proteins like those involved in the integrated stress response (ISR) pathway, including PHGDH, ASNS, and MTHFD1L, which may indicate underlying mitochondrial dysfunction despite normal expression levels of FAM136A interactors .

• Age-dependent effects: The phenotypes associated with FAM136A dysfunction, particularly in mouse models, may be age-dependent, requiring longitudinal studies to fully capture expression changes and functional consequences .

• Validation across platforms: Employ multiple detection methods (qPCR, Western blot, immunohistochemistry) and different antibodies to confirm expression patterns.

How is FAM136A immunoreactivity associated with lung cancer prognosis and what methodologies should researchers use for assessment?

FAM136A immunoreactivity has significant clinical associations in lung cancer research. In a study of 177 lung carcinoma tissues, FAM136A immunoreactivity was detected in 44.6% (79/177) of cases and showed significant associations with:

• Tumor T stage (P = 0.035)

• Lymph node metastasis (P = 0.046)

• TNM staging system classification

For researchers investigating FAM136A as a prognostic marker in cancer:

• Antibody selection: Use validated antibodies at appropriate dilutions (1:50 for immunohistochemistry has been reported in lung cancer studies) .

• Scoring system: Implement a standardized semi-quantitative scoring system for extent of immunoreaction:

  • 0: 0% immunoreactive cells

  • 1: <5% immunoreactive cells

  • 2: 5-50% immunoreactive cells

  • 3: >50% immunoreactive cells

• Statistical analysis: Correlate FAM136A status with clinicopathological parameters using appropriate statistical tests (chi-square test for categorical variables, Kaplan-Meier method with log-rank test for survival analysis) .

• Subgroup analysis: Consider analyzing FAM136A expression specifically in patient subgroups (e.g., those with lymph node metastasis) to identify cohort-specific prognostic value .

What experimental strategies should researchers employ to investigate FAM136A's role in Ménière's disease pathophysiology?

To investigate FAM136A's contribution to Ménière's disease (MD) pathophysiology, researchers should consider a multi-level experimental approach:

• Patient-derived cell models: Obtain lymphoblastoid cell lines (LCLs) from MD patients with FAM136A mutations (such as the Q76* mutation) for functional studies .

• Rescue experiments: Reintroduce wild-type FAM136A into patient-derived cells and measure the recovery of:

  • IMS proteostasis markers (HAX1, CLPB, ENDOG)

  • OXPHOS protein levels

  • Mitochondrial respiratory function

• Mouse models: Utilize existing Fam136a knockout mice that present with age-related hearing loss reminiscent of Ménière's disease for:

  • Age-dependent phenotypic characterization

  • Tissue-specific proteomics (particularly inner ear tissues)

  • Assessment of integrated stress response activation

• Investigate connections with other MD-associated genes: Particularly focus on HAX1 and CLPB, two MD-associated genes that show co-essentiality patterns with FAM136A and are depleted in FAM136A-deficient cells .

• Heterozygous vs. homozygous model comparisons: Since MD patients typically carry heterozygous FAM136A mutations with autosomal-dominant inheritance, but research models often use homozygous inactivation, carefully design experiments to compare gene dosage effects .

What controls should researchers include when using FAM136A antibodies for experimental validation?

To ensure robust and reproducible results when using FAM136A antibodies, researchers should implement the following controls:

• Negative controls:

  • Primary antibody omission: Replace primary antibody with the antibody diluent (e.g., PBS)

  • Isotype control: Use matched concentration of non-specific IgG from the same species

  • Tissue negative controls: Include tissues known not to express FAM136A or with very low expression

  • siRNA/CRISPR knockdown: Generate FAM136A-depleted samples as specificity controls

• Positive controls:

  • Validated cell lines: U2OS, A549, HeLa, Jurkat, or MCF-7 cells have confirmed FAM136A expression

  • Tissue positive controls: Mouse testis tissue or human ovarian cancer tissue have been validated

  • Overexpression system: Cells transfected with FAM136A expression construct

• Technical validation:

  • Western blot: Confirm antibody recognizes a band at the expected molecular weight (15-16 kDa)

  • Multiple antibodies: Use antibodies targeting different epitopes of FAM136A when possible

  • Multiple applications: Confirm specificity across different techniques (WB, IHC, IF)

• Additional controls for IHC/IF:

  • Autofluorescence control: Examine unstained sections to assess background

  • Secondary antibody only: Control for non-specific binding of secondary antibody

How can researchers optimize FAM136A antibody-based detection in co-localization studies with mitochondrial markers?

For successful co-localization studies investigating FAM136A's mitochondrial localization:

• Fixation optimization:

  • Test both paraformaldehyde (4%) and methanol fixation, as mitochondrial proteins can show different accessibility depending on fixation method

  • Consider mild permeabilization protocols that preserve mitochondrial structure

• Mitochondrial marker selection:

  • Outer membrane: TOM20 or VDAC

  • Inner membrane: TIM23 or Complex V subunits

  • Matrix: HSP60 or mitochondrial HSP70

  • IMS-specific: SMAC/DIABLO or Cytochrome c

  • For FAM136A specifically, co-staining with IMS markers would be most informative based on its localization

• Microscopy considerations:

  • Super-resolution techniques (STED, SIM, PALM/STORM) are preferable for distinguishing submitochondrial compartments

  • Z-stack acquisition with deconvolution to improve spatial resolution

  • Careful channel alignment and bleed-through controls

• Quantitative co-localization:

  • Use established co-localization coefficients (Pearson's, Manders')

  • Implement object-based co-localization for punctate signals

  • Set thresholds based on control samples

• Validation approaches:

  • Biochemical fractionation to confirm FAM136A enrichment in mitochondrial fractions

  • Protease protection assays to confirm submitochondrial localization

  • Immuno-electron microscopy for highest resolution localization

What are the most common technical challenges when working with FAM136A antibodies and how can researchers address them?

Based on the available research on FAM136A antibodies, researchers should anticipate and address these common technical challenges:

• Specificity concerns:

  • Challenge: Cross-reactivity with related proteins

  • Solution: Validate using FAM136A knockout/knockdown controls; confirm expected molecular weight (15-16 kDa) ; use multiple antibodies targeting different epitopes

• Subcellular localization ambiguity:

  • Challenge: Distinguishing cytoplasmic from mitochondrial signal

  • Solution: Perform subcellular fractionation; use high-resolution imaging with mitochondrial markers; compare multiple fixation protocols which may differentially preserve compartmentalization

• Inter-tissue variability:

  • Challenge: Inconsistent detection across tissue types

  • Solution: Optimize antigen retrieval methods per tissue; consider tissue-specific expression levels; adjust antibody concentration accordingly (1:50-1:500 for IHC)

• Quantification challenges:

  • Challenge: Semi-quantitative assessment of staining intensity

  • Solution: Implement standardized scoring system ; use digital image analysis when possible; include reference standards on each slide

• Epitope masking in disease states:

  • Challenge: Altered detection in pathological samples

  • Solution: Test multiple antibodies recognizing different epitopes; compare multiple antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

How might FAM136A antibodies be utilized in studying integrated stress response pathways in mitochondrial diseases?

FAM136A depletion has been associated with activation of the integrated stress response (ISR) in mouse tissues, suggesting a promising avenue for investigating mitochondrial dysfunction . Researchers interested in exploiting FAM136A antibodies for ISR studies should consider:

• Multiplex immunostaining protocols:

  • Combine FAM136A antibodies with ISR markers (PHGDH, ASNS, MTHFD1L)

  • Implement sequential immunofluorescence or spectral unmixing for co-detection

  • Quantify correlation between FAM136A levels and ISR activation strength

• Tissue-specific analysis strategies:

  • Focus on tissues showing strong ISR activation upon FAM136A depletion (heart and liver in mouse models)

  • Compare with tissues that maintain FAM136A function despite mutation (potential compensatory mechanisms)

  • Investigate age-dependent changes in this relationship

• Therapeutic intervention monitoring:

  • Use FAM136A antibodies to track restoration of mitochondrial function following ISR-targeting interventions

  • Monitor changes in both FAM136A levels and its interacting partners (HAX1, CLPB) during treatment

• Patient stratification biomarker development:

  • Explore FAM136A immunodetection as a potential biomarker for selecting patients likely to respond to mitochondrial or ISR-targeting therapies

  • Develop standardized immunohistochemical protocols applicable to clinical samples

What experimental approaches should researchers consider when investigating conflicting FAM136A expression data across cancer types?

FAM136A shows variable expression and functional relationships across cancer types, requiring careful experimental design to address apparent conflicts in expression data:

• Cancer cell line dependency analysis:

  • Leverage public databases like the Cancer Cell Line Encyclopedia (CCLE) to examine FAM136A dependency profiles across >1,000 cancer cell lines

  • Design experiments targeting both FAM136A-dependent and independent cell types

  • Correlate dependency with genomic features (mutations, CNVs, expression profiles)

• Co-essentiality network analysis:

  • Identify genes showing similar dependency patterns as FAM136A across cancer cell lines

  • Focus on pathways such as "intramitochondrial membrane organization," "protein import, sorting, and homeostasis," and "OXPHOS"

  • Design validation experiments for key predicted functional interactions

• Comparative tissue microarray studies:

  • Develop standardized IHC protocols (1:50-1:500 dilution range)

  • Implement consistent scoring systems for extent of immunoreaction

  • Compare FAM136A expression across multiple cancer types simultaneously

• Integration with patient outcome data:

  • Stratify analysis based on cancer subtype, stage, and therapy response

  • Compare FAM136A expression with established biomarkers

  • Particularly focus on cancers with frequent lymph node involvement, as FAM136A has shown prognostic value in this context