TTC17 Antibody

What is TTC17 and what are its known functional roles in cells?

TTC17 is a large (~130-170 kDa) protein containing tetratricopeptide repeat domains that plays multiple roles in cellular function. Recent research has identified TTC17 as:

• An endoplasmic reticulum (ER) resident protein involved in protein folding, quality control, and trafficking within the secretory pathway

• A modulator of actin polymerization with a role in primary ciliogenesis

• A potential metastasis suppressor in breast cancer through regulation of the RAP1/CDC42 pathway

TTC17 is approximately 136 kb long, located in bands 12–11.2 on the short arm of chromosome 11 (11p12-p11.2), and comprises 27 exons . It contains two sets of TPR domains - one with three repeats and another with four repeats - that likely facilitate protein-protein interactions .

What are the optimal conditions for using TTC17 antibodies in immunohistochemistry?

For immunohistochemistry applications with TTC17 antibodies, consider the following optimization parameters:

To ensure optimal staining, it is recommended to:

• Perform antigen retrieval by heat-induced epitope retrieval methods

• Include positive control tissues (e.g., human kidney) and negative controls

• Titrate the antibody concentration for each specific tissue type being examined

How can I validate the specificity of a commercial TTC17 antibody?

Validating antibody specificity is crucial for reliable experimental outcomes. For TTC17 antibodies, consider these validation approaches:

• CRISPR/Cas9 knockout validation: Generate TTC17 knockout cell lines using CRISPR/Cas9 technology targeting sequences like 5′-CACGCACTGGGTCGTCACGG-3′ as used in published research . The absence of signal in knockout cells compared to wild-type confirms antibody specificity.

• Molecular weight verification: TTC17 should appear at approximately 170 kDa on Western blots, with potential shifts to ~144 kDa after PNGaseF treatment due to removal of N-glycans .

• Multiple antibody comparison: Use antibodies targeting different epitopes of TTC17 and compare staining patterns.

• Transfection-based validation: Overexpress tagged versions of TTC17 (e.g., FLAG-tagged) and confirm co-localization with antibody staining .

• RNA interference: Perform siRNA knockdown of TTC17 and verify reduced antibody signal proportional to mRNA reduction.

Which TTC17 isoforms are most commonly expressed, and how does this affect antibody selection?

Research has identified multiple TTC17 isoforms, with isoform-specific expression patterns that should guide antibody selection:

For antibody selection:

• Choose antibodies targeting the N-terminal region (amino acids 20-250) for detecting multiple isoforms

• For isoform-specific detection, select antibodies targeting unique regions of each isoform

• When studying cell lines, verify which isoform predominates in your specific model system

Notably, transcriptome analysis demonstrates that isoform expression varies across tissue types , suggesting that researchers should verify isoform expression in their specific experimental system before selecting antibodies.

What are the recommended protocols for using TTC17 antibodies in Western blotting?

For optimal Western blot detection of TTC17:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Include glycosidase inhibitors if glycosylation status is important

• Gel electrophoresis:

  • 6-8% SDS-PAGE gels are recommended for the large TTC17 protein

  • Include PNGaseF-treated controls to verify glycosylation-dependent migration

• Antibody dilutions:

  • Primary antibody: 1/500 - 1/2000

  • Secondary antibody: Typically 1/5000 - 1/10000 (HRP-conjugated)

• Controls to include:

  • Positive control (e.g., HEK293 cells express detectable levels of TTC17)

  • CRISPR knockout cell lysate as negative control

  • Size marker to verify ~170 kDa band

• Special considerations:

  • Extended transfer time (90-120 minutes) may be necessary for complete transfer of this large protein

  • Blocking with 5% non-fat milk is typically effective

How can TTC17 antibodies be used to investigate its role in breast cancer metastasis?

TTC17 has been identified as a potential metastasis suppressor in breast cancer, making it an important target for cancer research. Methodological approaches include:

• Tissue microarray analysis:

  • Use TTC17 antibodies for IHC staining of breast cancer tissue microarrays

  • Calculate H-scores (0-300 range) by combining signal intensity and proportion of positively stained cells

  • Compare expression between primary tumors and metastatic sites

• Correlation with clinical parameters:

  • Examine TTC17 expression in relation to:

    • Lymph node status (N0 vs N2/N3)

    • Tumor size (T1/T2 vs T3/T4)

    • Patient survival data

  • Published data indicates that low TTC17 expression correlates with more aggressive clinicopathological characteristics

• Mechanistic studies:

  • Use TTC17 antibodies with RAP1/CDC42 pathway markers in co-immunostaining to explore regulatory relationships

  • Combine with phospho-specific antibodies to evaluate signaling activity

• Therapeutic response prediction:

  • Research indicates TTC17-silenced breast cancer cells show enhanced sensitivity to rapamycin and paclitaxel

  • Use antibodies to stratify patient samples for correlation with treatment response

What approaches can resolve detection challenges when studying TTC17's glycosylation states?

TTC17 is heavily glycosylated with up to 12 predicted N-linked glycosylation sites, presenting unique detection challenges:

• Enzymatic deglycosylation strategy:

  • PNGaseF treatment results in a significant (~26 kDa) mobility shift, confirming extensive glycosylation

  • EndoH treatment helps distinguish ER-resident (sensitive) vs. Golgi-processed (resistant) forms

• Site-specific glycosylation mapping:

  • Use TTC17 antibodies for immunoprecipitation followed by LC-MS/MS analysis

  • PNGaseF treatment converts glycosylated Asn to Asp, creating a mass shift detectable by MS

  • Eight glycosylation sites have been confirmed using this approach

• Antibody selection for glycoform detection:

  • Choose epitopes away from glycosylation sites to avoid glycan-dependent recognition issues

  • Consider generating glycoform-specific antibodies for specialized applications

• Visualizing glycoform distribution:

  • Combine TTC17 antibodies with lectins to visualize specific glycan structures

  • Use 2D gel electrophoresis to separate glycoforms prior to immunoblotting

How can TTC17 antibodies be applied in studying protein trafficking within the secretory pathway?

TTC17 functions as a trafficking factor in the secretory pathway, and antibodies can help elucidate this role:

• Proximity labeling approaches:

  • Generate TTC17-BioID or TTC17-APEX fusion proteins

  • Use TTC17 antibodies to verify expression and localization

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

• Co-immunoprecipitation studies:

  • TTC17 interacts with various chaperones and co-chaperones in the ER

  • Use antibodies for immunoprecipitation followed by identification of binding partners

• Comparative secretome analysis:

  • Use TTC17 antibodies to verify knockout/knockdown efficiency

  • Quantitative proteomics comparing WT vs. TTC17-/- cells has identified over 300 proteins with altered trafficking

  • Notable trafficking changes include GPNMB (8.8-fold enriched), clusterin (3.5-fold), and UGGT1 (1.63-fold)

• Subcellular fractionation:

  • ER and Golgi fractionation followed by immunoblotting with TTC17 antibodies

  • Combine with organelle markers to track localization

What are common technical challenges when using TTC17 antibodies and how can they be addressed?

How should experimental design account for stress-induced changes in TTC17 expression?

TTC17 expression is significantly upregulated under various ER stress conditions, which must be considered in experimental design:

• Documented stress responses:

  • Tunicamycin treatment: 18.1-fold increase in TTC17 protein levels

  • Brefeldin A treatment: 6.9-fold increase in TTC17 protein levels

• Experimental considerations:

  • Include unstressed controls in all experiments

  • Consider time-course experiments to track expression changes

  • Monitor both protein and mRNA levels, as transcriptional upregulation occurs during stress

• Stress markers to include:

  • Co-stain with BiP/GRP78, CHOP, or XBP1s as ER stress markers

  • Use ATF6 activation as an additional stress indicator

• Normalization strategy:

  • Select housekeeping genes/proteins that remain stable under ER stress conditions

  • Consider using total protein normalization instead of single reference proteins

What strategies can resolve contradictory findings about TTC17 subcellular localization?

The literature contains some contradictions regarding TTC17 localization:

• Methodological reconciliation:

  • Use multiple antibodies targeting different epitopes

  • Combine with epitope-tagged constructs for validation

  • Perform co-localization with established markers for different compartments

• Key experimental evidence:

  • EndoH sensitivity supports predominant ER localization

  • Signal sequence and N-glycosylation confirm secretory pathway targeting

  • Co-localization with ER markers provides further confirmation

• Technical approaches:

  • Super-resolution microscopy to resolve fine localizations

  • Subcellular fractionation with immunoblotting

  • Electron microscopy with immunogold labeling for highest resolution

• Addressing contradictions:

  • Some reports describe TTC17 as cytoskeletal or Golgi-localized

  • These differences may reflect cell-type specific localization, isoform differences, or technical limitations

  • Sequential detergent extraction can help distinguish between cytoskeletal, membrane-bound, and soluble pools

How might TTC17 antibodies contribute to understanding cancer drug sensitivity mechanisms?

Recent research has uncovered potential connections between TTC17 expression and drug sensitivity:

• Drug sensitivity correlations:

  • TTC17-silenced breast cancer cells show enhanced sensitivity to rapamycin and paclitaxel

  • This suggests potential for TTC17 as a stratification marker for treatment selection

• Methodological approaches:

  • Use TTC17 antibodies for IHC on patient tissues before and after treatment

  • Correlate expression with treatment response metrics

  • Combine with phospho-specific antibodies to track associated signaling pathways (e.g., RAP1/CDC42)

• Experimental models:

  • Patient-derived xenografts stratified by TTC17 expression

  • Drug sensitivity testing in isogenic cell lines with/without TTC17

  • High-throughput drug screening with TTC17 expression as a variable

• Clinical correlation:

  • Retrospective analysis of TTC17 expression in patient samples from clinical trials

  • The TCGA database includes 1083 breast cancer patients with TTC17 expression data available for analysis

What considerations are important when using TTC17 antibodies across different model organisms?

When working across species:

• Verify the epitope sequence conservation between species

• Include appropriate positive and negative controls from each species

• Optimize antibody concentration for each species separately

• Consider generating species-specific antibodies for critical applications

TTC17 knockout mouse models have been used in cancer metastasis studies , making cross-reactive antibodies valuable for translational research between mouse models and human samples.