TMEM109 Antibody

What is the optimal application for detecting TMEM109 in tissue samples?

Western blotting represents the most validated application for TMEM109 detection, though immunohistochemistry (IHC) and immunofluorescence (IF) are also viable depending on experimental goals. For Western blot applications, most commercially available antibodies perform optimally at dilutions between 1:2,000 and 1:10,000 . For immunohistochemistry applications, recommended dilutions typically range from 1:500 to 1:1000 for paraffin-embedded sections . When selecting between applications, consider:

When transitioning between applications, optimization of antibody concentration is essential for each specific tissue type and fixation method .

How can I validate TMEM109 antibody specificity for my experiments?

Methodological validation should include multiple complementary approaches:

• Positive control selection: Use tissues/cells known to express TMEM109 abundantly. Western blot detection should reveal a primary band at approximately 26.2 kDa .

• Knockout/knockdown validation: Compare antibody staining between wild-type samples and those with TMEM109 knockdown or knockout.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to demonstrate signal specificity.

• Cross-application validation: Confirm TMEM109 detection across multiple techniques (e.g., if using for IHC, validate with Western blot) .

• Epitope-specific validation: Select antibodies targeting different regions of TMEM109 (N-terminal, middle region, C-terminal) to confirm consistent localization patterns .

Several commercial antibodies have been extensively validated, with products from Atlas Antibodies (HPA011785), LSBio (LS-C817456), and Novus Biologicals (NBP1-93462) showing strong validation profiles across multiple applications .

What are the optimal storage and handling conditions for TMEM109 antibodies?

Storage and handling protocols significantly impact antibody performance and longevity:

Storage recommendations:

• Store at 4°C for short-term use (1-2 weeks)

• Store at -20°C for long-term preservation

• For lyophilized antibodies, reconstitute in distilled water and store in aliquots at -20°C

• For liquid formulations containing sodium azide preservative, minimize freeze-thaw cycles

Handling protocols:

• Always centrifuge briefly before opening vials to collect liquid at the bottom

• For glycerol-containing formulations (typically 40-50%), mix gently before use

• Working dilutions should be prepared immediately before use and not stored long-term

• When using biotinylated or fluorophore-conjugated antibodies, protect from light during all handling steps

Experimental evidence indicates that TMEM109 antibodies retain full activity for at least one year from receipt when stored according to manufacturer recommendations .

How should TMEM109 antibody protocols be optimized for Western blot applications?

For optimal Western blot results with TMEM109 antibodies, implement this methodological approach:

• Sample preparation: Use RIPA or NP-40 buffer with protease inhibitors. Heat samples at 95°C for 5 minutes in reducing sample buffer.

• Gel selection: 12-15% polyacrylamide gels provide optimal resolution for the 26.2 kDa TMEM109 protein .

• Blocking optimization: 5% skim milk in PBS buffer typically provides optimal blocking for TMEM109 detection .

• Antibody dilution: Start with a 1:5,000 dilution for most commercial antibodies, then optimize. Most protocols recommend using antibody concentrations around 1.25 μg/mL .

• Secondary antibody selection: HRP-conjugated anti-rabbit IgG at 1:50,000-1:100,000 dilution is optimal for most TMEM109 primary antibodies .

• Membrane washing: Implement stringent washing (4-5 times for 5 minutes each) with PBST between antibody incubations to reduce background.

• Signal development: Use enhanced chemiluminescence with exposure times starting at 30 seconds and adjusting as needed.

For membrane stripping and re-probing, mild stripping buffers are preferred to preserve epitope integrity when working with TMEM109.

What are the key considerations when selecting TMEM109 antibodies for cross-species studies?

Species cross-reactivity is a crucial consideration for comparative studies, requiring careful antibody selection:

When designing cross-species experiments:

• Select antibodies raised against conserved epitopes between target species

• Validate antibodies separately in each species before comparative studies

• Adjust antibody concentration for each species, as optimal dilutions may differ

• Consider using antibodies targeting the middle region (AA 101-150) of TMEM109 for maximum cross-reactivity

• Perform BLAST analysis of the immunogen sequence against target species to predict reactivity

For difficult-to-detect species orthologs, custom antibody development against species-specific sequences may be necessary.

How can researchers accurately quantify TMEM109 expression levels across different experimental conditions?

For precise quantification of TMEM109 expression:

• Normalization strategy: When quantifying TMEM109 by Western blot, normalize against:

  • Housekeeping proteins (β-actin, GAPDH) for total cell expression

  • Compartment-specific markers (Lamin B1 for nuclear fraction, Calnexin for ER fraction) when studying subcellular distribution

• Quantitative techniques:

  • Western blot with densitometry (semi-quantitative)

  • ELISA for more precise quantification (multiple antibodies available with ELISA validation)

  • RT-qPCR in parallel to correlate protein and mRNA expression

• Standards and controls:

  • Include concentration gradients of recombinant TMEM109 protein as standard curve

  • Process all experimental conditions simultaneously with identical antibody concentrations and development times

• Statistical analysis:

  • Use at least three biological replicates for meaningful quantification

  • Apply appropriate statistical tests based on data distribution

• Environmental factors affecting expression:

  • Consider that chemicals like tetrachlorodibenzodioxine, diuron, and thiram have been documented to decrease TMEM109 expression

  • UV exposure may alter expression due to TMEM109's role in UV damage response

How can TMEM109 antibodies be employed in cancer biomarker research?

Research demonstrates TMEM109's potential as a cancer biomarker, particularly in pancreatic cancer differentiation:

• Cancer type differentiation: In a study of 195 cases, TMEM109 immunostaining (combined with KRT17, ANXA10, and PTMS) successfully differentiated pancreatic head cancer (PHC) from distal cholangiocarcinoma (DCC) . The study found:

  • TMEM109 positivity in 75% of PHC cases vs. 42% of DCC cases in the initial cohort

  • In a larger validation cohort, TMEM109 positivity was observed in 66.7% of PHC cases compared to 35.4% of DCC cases (p=0.0003)

• Methodological approach for cancer studies:

  • Use standardized immunohistochemical protocols with consistent cutoffs (>10% tumor cell staining recommended)

  • Evaluate cytoplasmic staining patterns for TMEM109

  • Implement multi-marker panels rather than single markers

  • Consider using tissue microarrays for high-throughput screening

• Technical considerations for cancer tissue analysis:

  • Adjust antibody concentrations for formalin-fixed paraffin-embedded tissues

  • Include appropriate tissue controls in each staining batch

  • Consider double immunostaining with cell-type specific markers to identify expressing cell populations

This evidence supports TMEM109's potential utility in diagnostic pathology applications, particularly when combined with other biomarkers.

What is the role of TMEM109 in DNA damage response, and how can antibodies help elucidate this function?

TMEM109 is implicated in DNA damage response pathways, particularly in protection against ultraviolet C-induced cell death . Researchers investigating this function should consider:

• Experimental design for UV damage studies:

  • Compare TMEM109 localization before and after UV exposure using immunofluorescence

  • Perform co-immunoprecipitation with TMEM109 antibodies to identify interaction partners in DNA damage response

  • Use subcellular fractionation followed by Western blotting to track TMEM109 translocation between ER and nucleus following damage

• Protein-protein interaction studies:

  • TMEM109 has documented interactions with UBC and Rassf1

  • Immunoprecipitation protocols should be optimized using approximately 20 μL antibody per mg of cell lysate

  • Consider crosslinking approaches to capture transient interactions

• Signaling pathway analysis:

  • TMEM109 is predicted to be involved in cellular response to gamma radiation

  • May participate in intrinsic apoptotic signaling pathways in response to DNA damage mediated by p53

  • Investigate phosphorylation status using phospho-specific antibodies if available

• Chemical modulation:

  • Numerous compounds affect TMEM109 expression, including doxorubicin, tetrachloromethane, and thioacetamide

  • These can be employed as experimental tools to modulate TMEM109 levels

For comprehensive DNA damage response studies, pair TMEM109 antibody approaches with functional assays like comet assays, γH2AX foci quantification, and cell survival measurements.

How should researchers approach contradictory results when using different TMEM109 antibodies?

When confronted with contradictory results between different TMEM109 antibodies, implement this systematic troubleshooting approach:

• Epitope mapping analysis:

  • Compare the exact epitopes targeted by each antibody (N-terminal, middle region, C-terminal)

  • Different antibodies may detect distinct isoforms or post-translationally modified forms

  • Antibodies targeting amino acids 25-75 , 34-83 , and 101-150 regions may yield different results

• Validation hierarchy:

  • Prioritize results from antibodies with the most extensive validation evidence

  • Consider the validation status in specific applications (IHC vs. WB vs. IF)

  • Antibodies with published research citations should be given greater weight

• Technical reconciliation:

  • Optimize conditions separately for each antibody

  • Test multiple antibody concentrations and incubation conditions

  • Evaluate fixation-sensitive epitopes that may be masked in certain preparations

• Orthogonal validation:

  • Confirm findings using non-antibody methods (mRNA expression, genetic tagging)

  • Apply genetic approaches (siRNA knockdown, CRISPR knockout) to verify specificity

  • Consider mass spectrometry validation of immunoprecipitated proteins

When publishing results with TMEM109 antibodies, transparent reporting of antibody catalog numbers, dilutions, and complete protocols is essential for reproducibility.

How can TMEM109 antibodies be adapted for high-throughput or automated screening platforms?

For integration into high-throughput screening platforms:

• Antibody format selection:

  • Biotin-conjugated antibodies facilitate automated detection systems

  • Fluorophore-conjugated antibodies (FITC, PE, APC) enable direct detection without secondary antibodies

  • HRP-conjugated formats are compatible with automated ELISA platforms

• Protocol optimization for automation:

  • Minimize incubation steps and washing cycles

  • Standardize antibody concentrations to reduce batch variation

  • Develop robust positive and negative controls for each plate/run

• Multiplexing strategies:

  • Combine TMEM109 detection with other biomarkers (e.g., KRT17, ANXA10, PTMS in pancreatic cancer applications)

  • Use differently labeled antibodies for simultaneous detection

  • Consider sequential staining protocols for antibodies from the same host species

• Data analysis approaches:

  • Implement machine learning algorithms for image analysis in IHC/IF applications

  • Develop standardized scoring systems specific to TMEM109 expression patterns

  • Create reference datasets for calibration across laboratories

These adaptations can facilitate TMEM109 analysis across large sample sets, such as tissue microarrays or multi-center clinical studies.

What considerations are important when developing functional studies of TMEM109 using antibody-based approaches?

When designing functional studies of TMEM109:

• Neutralizing antibody considerations:

  • Determine if commercially available antibodies possess neutralizing activity

  • Target antibodies to functional domains rather than just detection epitopes

  • Validate functional inhibition through complementary assays

• Live-cell applications:

  • Evaluate antibody internalization capacity if targeting cell-surface exposed regions

  • Consider generating single-chain antibody fragments for improved cellular penetration

  • Develop protocols for antibody delivery into living cells (e.g., protein transfection)

• Dynamic process monitoring:

  • Use fluorescently-labeled antibodies for tracking TMEM109 translocation or trafficking

  • Establish stable cell lines expressing tagged TMEM109 for live imaging

  • Combine with optogenetic approaches for precise temporal control

• Validation standards:

  • Include genetic manipulation controls (overexpression, knockout)

  • Perform domain-specific mutational analysis to map functional regions

  • Confirm antibody access to the relevant cellular compartments (nucleus, ER)

Research suggests TMEM109 mediates cellular responses to DNA damage , making functional antibody applications particularly valuable for investigating damage response pathways.