TMEM106B Antibody

What is TMEM106B and why is it significant in neurodegenerative research?

TMEM106B (transmembrane protein 106B) is a critical genetic risk factor for frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). Its significance extends to amyotrophic lateral sclerosis (ALS), which shares pathological TDP-43 inclusions with FTLD-TDP. Research indicates that TMEM106B expression levels in the brain may be directly linked to disease mechanisms in FTLD-TDP, with risk alleles potentially conferring genetic susceptibility by increasing gene expression. The protein can be detected at molecular weights ranging from 31-55 kDa and sometimes 70-90 kDa in experimental contexts, suggesting post-translational modifications or alternative forms of the protein . Understanding TMEM106B's biology is therefore essential for advancing our knowledge of multiple neurodegenerative conditions and their underlying pathological mechanisms.

What applications are validated for commercial TMEM106B antibodies?

TMEM106B antibodies have been validated for multiple laboratory applications across different experimental systems. According to published research, the most robustly validated applications include:

These applications have been documented in multiple peer-reviewed publications, with western blotting appearing in at least 6 publications and immunofluorescence in at least 4 publications according to current data . The optimal dilution should be determined empirically for each experimental system as sensitivity can vary based on sample type, protein expression levels, and specific detection methods employed.

What epitopes of TMEM106B are targeted by available antibodies?

Several antibodies targeting different epitopes of TMEM106B are available for research applications. The epitope specificity is critical for experimental design as it determines which forms of the protein will be detected. Key epitope regions include:

• N-terminal domain antibodies: Recognize the physiological form of TMEM106B

• C-terminal fragment (CTF) antibodies targeting specific residues:

  • Residues 140-163 (though antibodies to this region have shown low titers)

  • Residues 164-187

  • Residues 188-211 (shows strong immunohistochemical reactivity to CTF in aging and disease-associated brain)

  • Residues 239-250 (extensively detects TMEM-immunoreactive material)

  • Residues 253-274

The antibody recognizing residues 188-211 has been specifically highlighted for its effectiveness in immunohistochemical investigations of CTF accumulation in the brain . When designing experiments, researchers should select antibodies based on whether they wish to detect full-length TMEM106B, specific fragments, or pathological aggregates.

What antigen retrieval methods are optimal for TMEM106B immunohistochemistry?

Optimization of antigen retrieval is critical for successful TMEM106B immunohistochemistry, particularly when working with formalin-fixed, paraffin-embedded tissues. Research indicates that formic acid (FA) treatment is the most effective antigen retrieval agent for detecting TMEM106B C-terminal fragments in brain tissue. The recommended protocol includes:

• Deparaffinization and rehydration of 7-μm-thick paraffin sections

• Formic acid treatment for 1 minute (critical step)

• Washing in distilled water for 3 minutes

• Incubation with 3% hydrogen peroxide in PBS for 30 minutes to eliminate endogenous peroxidase activity

• Washing with PBS followed by blocking solution application for 20 minutes

• Primary antibody incubation overnight at 4°C

For some applications and epitopes, alternative antigen retrieval methods may be considered:

• TE buffer pH 9.0 (suggested for some applications)

• Citrate buffer pH 6.0 (alternative option)

Each antibody and tissue type may require specific optimization, so preliminary testing of multiple retrieval methods is advised for new experimental systems.

How should researchers validate TMEM106B antibody specificity?

Rigorous validation of TMEM106B antibody specificity is essential for producing reliable experimental results. Based on published methodologies, a comprehensive validation approach should include:

• Peptide adsorption tests: Pre-adsorb antibodies with the specific peptide immunogens used for their generation (e.g., 30 μg of synthetic peptide corresponding to target residues). Compare immunoreactivity between pre-adsorbed and non-adsorbed antibodies on identical sections to confirm specific binding.

• ELISA validation: Test antibody titers against the peptide immunogens to confirm recognition capability.

• Multiple antibody comparison: Use antibodies targeting different epitopes on adjacent tissue sections to confirm consistent staining patterns.

• Positive and negative control tissues: Include tissues known to express or lack TMEM106B (e.g., young cases typically lack TMEM-immunoreactive material).

• Quantitative assessment: Measure positive staining areas in standard microscopic fields using appropriate imaging software (e.g., cellSens Dimension Desktop) .

This multi-faceted approach provides strong evidence for antibody specificity and ensures that observed immunoreactivity represents genuine TMEM106B detection.

What expression patterns of TMEM106B are observed in control versus disease tissues?

TMEM106B expression and immunoreactivity patterns differ significantly between control and disease tissues, particularly in age-dependent and disease-specific contexts:

• Age-dependent patterns:

  • Young cases (20s): Typically lack significant TMEM-immunoreactive material

  • Elderly subjects (>65 years): Variable TMEM-immunoreactive material accumulation, even without neurodegenerative disease diagnosis

• Disease-specific patterns:

  • Frontotemporal lobar degeneration with motor neuron disease: Often shows significant TMEM-immunoreactive material

  • Dementia with Lewy bodies: Can exhibit abundant TMEM-immunoreactive material

  • Multiple system atrophy: May display TMEM-immunoreactive material accumulation

• Cellular localization:

  • TMEM-immunoreactive material appears in the cytoplasm of various cell types in the frontal lobe

  • Double-label immunofluorescence studies show TMEM-immunoreactive material accumulates without colocalization with other pathogenic proteins

When designing studies, researchers should include appropriate age-matched controls and recognize that TMEM106B accumulation occurs in aging even without neurodegenerative disease diagnosis, requiring careful interpretation of results.

How do different TMEM106B antibodies compare in detecting pathological aggregates?

Different TMEM106B antibodies vary significantly in their ability to detect pathological aggregates, particularly in aging and disease-associated brain tissue. Comparative research has revealed:

• Antibody targeting residues 239-250: Demonstrates extensive detection capabilities for TMEM-immunoreactive material in individuals with TMEM106B fibril accumulation. This antibody has been used as a reference standard to identify cases with abundant TMEM106B pathology.

• Antibody targeting residues 188-211: Shows significant affinity for TMEM-immunoreactive material, with larger positive areas in TMEM-immunoreactive material-positive cases compared to negative cases. This antibody represents an important alternative for detecting TMEM106B CTF accumulation.

• Antibodies targeting residues 164-187 and 253-274: These showed different immunostaining patterns compared to the 239-250 and 188-211 antibodies, potentially identifying different forms or states of the protein.

• N-terminal antibodies: Typically recognize the physiological form of TMEM106B rather than pathological aggregates .

For studies specifically investigating pathological aggregates, antibodies targeting residues 239-250 or 188-211 are currently the most validated options. Researchers should consider using multiple antibodies targeting different epitopes to gain comprehensive insights into TMEM106B pathology.

What methodological approaches can resolve contradictory findings when using different TMEM106B antibodies?

When faced with contradictory findings using different TMEM106B antibodies, researchers should implement systematic troubleshooting approaches:

• Correlation analysis between antibodies: Evaluate the degree of concordance between areas positively stained by different antibodies. For example, selecting corresponding microscopic fields from consecutive thin paraffin sections and calculating Pearson correlation coefficients between immunoreactivities.

• Ultra-thin section analysis: Use consecutive 2.5-μm-thick paraffin sections with surfaces facing upward, stained with different antibodies, to minimize tissue variation effects.

• Quantitative comparison: Employ image analysis software to quantify positive areas in standard microscopic fields (e.g., 40× magnification covering 103,823 μm² or 10× magnification covering 1,661,174 μm²).

• Double-label immunofluorescence: When feasible, perform double-labeling with compatible antibodies to directly assess colocalization or divergence.

• Biochemical validation: Complement immunohistochemical findings with biochemical techniques such as western blotting to confirm molecular weight patterns and epitope accessibility .

These methodological approaches can help resolve apparent contradictions and provide a more comprehensive understanding of TMEM106B biology in different experimental contexts.

What is the significance of TMEM106B C-terminal fragments in neurodegenerative disease pathology?

The C-terminal fragments (CTFs) of TMEM106B have emerged as critical components in neurodegenerative disease pathology:

• Accumulation patterns: TMEM106B CTFs accumulate in aging brains and show increased presence in several neurodegenerative conditions, particularly frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and those caused by progranulin mutations.

• Disease specificity: While most prominently associated with FTLD-TDP, TMEM106B CTF accumulation has been observed in multiple neurodegenerative conditions including dementia with Lewy bodies and multiple system atrophy, suggesting a broader relevance across the neurodegenerative disease spectrum.

• Age-dependence: Accumulation of TMEM-immunoreactive material is consistently observed in elderly subjects (>65 years), even without diagnosed neurodegenerative disease, while being typically absent in young cases, indicating an age-dependent process that may contribute to vulnerability to neurodegeneration.

• Cellular distribution: TMEM-immunoreactive material appears in the cytoplasm of various cell types in the frontal lobe without colocalization with other pathogenic proteins, suggesting a distinct pathological process.

• Potential mechanisms: The presence of CTFs may represent incomplete degradation, altered processing, or pathological aggregation of TMEM106B, potentially linked to lysosomal dysfunction that is increasingly recognized in neurodegenerative diseases .

Understanding the formation, accumulation, and impact of these CTFs may provide critical insights into disease mechanisms and potentially identify new therapeutic targets for multiple neurodegenerative conditions.

What are the critical storage and handling conditions for TMEM106B antibodies?

Proper storage and handling of TMEM106B antibodies are essential for maintaining reactivity and experimental reproducibility:

Researchers should carefully follow manufacturer recommendations as variations may exist between specific antibody preparations. Additionally, when working with antibodies generated against synthetic peptides, special attention should be paid to ensure the synthetic peptides used for generation meet quality standards (confirmed by HPLC and mass spectrometry) .

How should researchers quantify TMEM106B immunoreactivity in tissue sections?

Accurate quantification of TMEM106B immunoreactivity requires standardized approaches that minimize bias and ensure reproducibility:

• Field selection protocol: Randomly select standard microscopic fields (e.g., ten 40× fields covering 103,823 μm² each) from each section to avoid selection bias.

• Imaging standardization: Maintain consistent microscope settings, including exposure time, gain, and threshold parameters across all samples being compared.

• Software-based quantification: Utilize specialized image analysis software (e.g., cellSens Dimension Desktop) to quantify positive areas based on color thresholding.

• Data representation: Express results as immunopositive area per field (μm²/total field area) for statistical analysis and comparison.

• Classification criteria: Establish clear criteria for classifying samples as "positive" or "negative" for TMEM-immunoreactive material based on quantitative thresholds.

• Statistical analysis: Apply appropriate statistical tests to evaluate differences between experimental groups, with error bars indicating standard error of the mean .

This systematic approach provides objective quantification of immunoreactivity patterns and facilitates meaningful comparisons between experimental conditions or subject groups.

What are the emerging applications of TMEM106B antibodies in biomarker development?

TMEM106B antibodies show promising potential in biomarker development for neurodegenerative diseases, with several emerging applications:

• Histopathological classification: TMEM106B immunoreactivity patterns may help classify subtypes of frontotemporal lobar degeneration and related disorders, potentially refining diagnostic criteria.

• Age-related pathology assessment: Given the consistent observation of TMEM-immunoreactive material in elderly subjects, these antibodies could serve as markers of age-related protein accumulation independent of clinical disease manifestation.

• Stratification tool: The variable accumulation of TMEM-immunoreactive material among patients with similar clinical diagnoses suggests potential for stratifying patients into distinct biological subgroups that might respond differently to targeted therapies.

• Preclinical detection: The presence of TMEM106B pathology in seemingly asymptomatic elderly individuals raises the possibility of identifying preclinical neurodegenerative processes before symptom onset.

• Therapeutic efficacy monitoring: As therapeutic approaches targeting protein aggregation or lysosomal function advance, TMEM106B antibodies could provide metrics to assess treatment efficacy in clearing pathological aggregates .

Further research correlating TMEM106B immunoreactivity patterns with clinical outcomes and genetic profiles will be essential to establish validated biomarker applications.

How might researchers optimize TMEM106B antibodies for novel applications beyond standard immunoassays?

Optimization of TMEM106B antibodies for novel applications requires innovative approaches beyond conventional immunoassays:

• Super-resolution microscopy applications: Adapting TMEM106B antibodies for techniques such as STORM or STED microscopy may reveal subcellular localization details not visible with conventional microscopy. This would require optimization of fluorophore conjugation and sample preparation to maintain epitope recognition while achieving single-molecule resolution.

• Live cell imaging: Developing membrane-permeable fluorescently tagged antibody fragments (Fabs) or single-chain variable fragments (scFvs) derived from TMEM106B antibodies could enable tracking of TMEM106B dynamics in living cells.

• Proximity labeling applications: Conjugating TMEM106B antibodies with enzymes like APEX2 or BioID could identify proteins in close proximity to TMEM106B in different cellular compartments, elucidating its interaction network.

• Flow cytometry optimization: Adapting TMEM106B antibodies for flow cytometry applications could enable quantitative assessment of TMEM106B expression in specific cell populations isolated from complex tissues.

• Therapeutically relevant modifications: Exploring antibody engineering approaches to enhance blood-brain barrier penetration or promote clearance of pathological TMEM106B aggregates could transition these research tools toward therapeutic applications .

These advanced applications would require rigorous validation and optimization but could substantially expand our understanding of TMEM106B biology and pathology.