TTC39B Antibody

What is TTC39B and why is it significant for research?

TTC39B (tetratricopeptide repeat domain 39B) is a protein that plays a crucial role in regulating high-density lipoprotein (HDL) cholesterol metabolism. Its significance stems from its function in promoting the ubiquitination and degradation of oxysterol receptors LXR (NR1H2 and NR1H3) . Research has shown that TTC39B deficiency stabilizes LXR, which reduces atherosclerosis and fatty liver disease, making it an important target for metabolic disease research . The protein contains three consecutive tetratricopeptide repeat (TPR) motifs, suggesting it functions as a scaffolding protein mediating the association of HDL-regulating proteins .

What are the structural characteristics of human TTC39B protein?

Human TTC39B has the following key structural characteristics:

• Canonical protein length: 682 amino acid residues

• Molecular mass: Approximately 77 kDa

• Contains three consecutive TPR motifs

• Main feature: Domain of unknown function 3808 (DUF3808), spanning the majority of the protein

• Gene location: Chromosome 9p22.3 on the minus strand

• Genomic DNA length: 136,517 bases

• mRNA length: 3,276 bases

• Structure: 39 introns and 20 exons

Up to seven different isoforms have been reported for this protein, suggesting complex post-transcriptional regulation .

How conserved is TTC39B across species?

TTC39B is highly conserved across multiple species, as shown in this orthology comparison table:

This high conservation suggests critical biological functions that have been maintained throughout evolution . The gene orthologs have also been reported in mouse, rat, bovine, frog, zebrafish, and chicken species, making cross-species studies feasible .

What criteria should be used to select the appropriate TTC39B antibody for specific applications?

When selecting a TTC39B antibody, researchers should consider:

• Target specificity: Determine whether the antibody recognizes specific isoforms or all known isoforms of TTC39B. Some antibodies may cross-react with other TTC39 family members (TTC39A and TTC39C) .

• Application compatibility: Verify whether the antibody has been validated for your specific application (WB, IHC, ELISA, etc.). For example, certain TTC39B antibodies perform well in Western Blot (1:500-1:1000 dilution) and Immunohistochemistry (1:50-1:500 dilution) .

• Species reactivity: Confirm reactivity with your species of interest. Many commercially available antibodies react with human and mouse TTC39B, but validation for other species may be limited .

• Epitope location: Consider whether the antibody targets the N-terminal, C-terminal, or middle region. This is particularly important if studying specific domains or if post-translational modifications might affect antibody binding.

• Validation data: Request and review validation data including positive control samples. For TTC39B, mouse testis tissue has been documented as a positive control for Western Blot, while human liver tissue works for immunohistochemistry .

How can I validate the specificity of a TTC39B antibody?

A robust validation strategy for TTC39B antibodies should include:

• Positive and negative controls:

  • Use tissues with known TTC39B expression (liver, small intestine) as positive controls

  • Include tissues with low or no expression as negative controls

  • Consider TTC39B knockout samples if available

• Multiple detection methods: Compare results across techniques (Western blot, IHC, IF) to confirm consistent detection patterns.

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

• siRNA knockdown: Perform siRNA knockdown of TTC39B to verify signal reduction.

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight (77 kDa for canonical form, though observed weights of 59 kDa and 67 kDa have been reported due to potential post-translational modifications) .

• Cross-reactivity assessment: Test for potential cross-reactivity with paralogs TTC39A and TTC39C, especially when using antibodies targeting conserved domains.

What are the optimal conditions for Western blot detection of TTC39B?

For optimal Western blot detection of TTC39B:

• Sample preparation:

  • For tissues: Use RIPA buffer supplemented with protease inhibitors

  • For cultured cells: Direct lysis in Laemmli buffer can improve detection

• Protein loading: Load 20-50 μg of total protein for most tissue samples; higher amounts may be required for tissues with lower expression.

• Gel percentage: Use 8-10% SDS-PAGE gels for optimal separation.

• Transfer conditions:

  • Semi-dry transfer: 15V for 45 minutes

  • Wet transfer: 100V for 60 minutes at 4°C

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

• Primary antibody:

  • Dilution range: 1:500-1:1000

  • Incubation: Overnight at 4°C

• Detection: The expected molecular weight is 77 kDa, but observed weights of 59 kDa and 67 kDa have been reported .

• Positive control: Mouse testis tissue has been validated as a reliable positive control .

• Troubleshooting: If multiple bands appear, consider isoform expression or potential degradation. TTC39B has up to 7 reported isoforms .

What protocols are recommended for immunohistochemical detection of TTC39B?

For effective immunohistochemical detection of TTC39B:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) sections (4-6 μm thickness)

  • Heat-induced epitope retrieval is essential

• Antigen retrieval:

  • Primary method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Blocking:

  • 3% hydrogen peroxide (10 minutes)

  • 5% normal serum from secondary antibody host (1 hour)

• Primary antibody:

  • Dilution range: 1:50-1:500

  • Incubation: Overnight at 4°C

• Visualization:

  • DAB chromogen for brightfield microscopy

  • Appropriate fluorophore-conjugated secondary antibody for fluorescence

• Positive controls:

  • Human liver tissue and liver cancer tissue have been validated

• Analysis considerations:

  • TTC39B typically shows cytoplasmic and membrane localization

  • Expression patterns differ between normal and pathological tissues

How can TTC39B antibodies be used in co-immunoprecipitation experiments?

For successful co-immunoprecipitation (Co-IP) of TTC39B and interacting proteins:

• Lysis buffer selection:

  • Use a gentle lysis buffer (e.g., 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol)

  • Include protease and phosphatase inhibitors

  • Avoid harsh detergents that might disrupt protein-protein interactions

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 30 minutes at 4°C

  • Remove beads to reduce non-specific binding

• Antibody binding:

  • Use 2-5 μg of TTC39B antibody per 500 μg of total protein

  • Incubate overnight at 4°C with gentle rotation

• Precipitation:

  • Add Protein A/G beads and incubate for 1-2 hours at 4°C

  • Perform 4-5 gentle washes with lysis buffer

• Elution and analysis:

  • Elute with SDS-PAGE sample buffer

  • Analyze by Western blot using antibodies against suspected interacting proteins

• Controls:

  • IgG control from the same species as the TTC39B antibody

  • Input sample (5-10% of starting material)

Research has shown that LXRα can be co-immunoprecipitated with TTC39B regardless of epitope tag size, indicating they coexist in the same protein complex . This is a critical interaction to validate when studying TTC39B's role in lipid metabolism.

How can TTC39B antibodies be used to investigate its role in HDL cholesterol metabolism?

To investigate TTC39B's role in HDL cholesterol metabolism:

• Tissue-specific expression analysis:

  • Use immunohistochemistry with TTC39B antibodies to quantify expression in liver and intestinal tissues in normal versus hyperlipidemic models

  • Compare expression patterns between wild-type and disease models using Western blot quantification

• LXR degradation assay:

  • Employ TTC39B antibodies in conjunction with LXR antibodies to monitor the inverse relationship between protein levels

  • In cell cultures, perform cycloheximide chase experiments to measure LXR turnover rates in the presence or absence of TTC39B

• Ubiquitination analysis:

  • Use TTC39B antibodies for immunoprecipitation followed by ubiquitin antibody detection

  • Data has shown significantly less polyubiquitinated LXRα relative to unmodified LXRα in T39-deficient livers compared to wild-type

• HDL metabolism pathway analysis:

  • Apply TTC39B antibodies in ChIP assays to investigate occupancy at LXR target gene promoters

  • Results have demonstrated increased occupancy by LXR/RXR over LXREs of several LXR targets in TTC39B-deficient versus wild-type hepatocytes

• Therapeutic intervention studies:

  • Utilize TTC39B antibodies to monitor protein expression changes following experimental treatments targeting HDL metabolism

  • Compare with other metabolic markers to establish pathway relationships

What are the recommended approaches for studying TTC39B's role in fatty liver disease?

For investigating TTC39B's involvement in fatty liver disease:

• Histopathological analysis:

  • Use immunohistochemistry with TTC39B antibodies on liver sections from various stages of fatty liver disease

  • Correlate TTC39B expression with Oil Red O staining intensity, which indicates lipid accumulation

  • Research has shown that TTC39B-deficient mice exhibit less Oil Red O staining in liver tissues

• Molecular profiling:

  • Combine TTC39B antibody detection with analysis of:

    • Lipogenic gene expression (Scd1, Elovl5, Accα, Gpat1, G6pd)

    • LXR target genes (Srebf1, Abcg5, Abcg8)

    • Inflammatory markers

• Triglyceride synthesis assessment:

  • Study has demonstrated significantly decreased hepatic TG synthesis in TTC39B-deficient mice, accompanied by decreased expression of lipogenic genes

  • Use TTC39B antibodies to correlate protein levels with metabolic changes

• Liver-specific knockout models:

  • Mortality studies in tissue-specific TTC39B knockout mice revealed that protection from fatty liver disease was entirely due to hepatic TTC39B deficiency

  • Use antibodies to confirm knockout efficiency and study compensatory mechanisms

• Fibrosis and inflammation markers:

  • Research indicates TTC39B deficiency results in less perisinusoidal and periportal fibrosis, reduced inflammation, and less hepatocellular ballooning

  • Correlate these markers with TTC39B expression in various experimental models

What technical considerations should be addressed when using TTC39B antibodies to study protein-protein interactions?

When studying TTC39B protein-protein interactions:

• Buffer optimization:

  • Test multiple lysis conditions to preserve interactions

  • Consider crosslinking agents for transient interactions

  • Include protease inhibitors to prevent degradation of interaction partners

• Antibody epitope considerations:

  • Select antibodies whose epitopes do not interfere with known or suspected protein-binding domains

  • The immunogen sequence "SSSSTKVDLKSGLEECAVALNLFLSNKFTDALELLRPWAKESMYHALGYSTIVVLQAVLTFEQQDIQNGISAMKDALQ" has been validated for some antibodies

• Binding partner validation strategies:

  • Reverse co-immunoprecipitation using antibodies against suspected interaction partners

  • Proximity ligation assay to visualize interactions in situ

  • Mass spectrometry analysis of immunoprecipitated complexes

• LXR interaction studies:

  • Research has shown that endogenous LXR can be isolated using immobilized GW3965 (LXR agonist)

  • LXRα co-immunoprecipitated with TTC39B regardless of epitope tag size

• Functional validation:

  • Proteasome inhibitor studies (e.g., bortezomib) have shown differential effects on LXR levels in wild-type versus TTC39B-deficient hepatocytes

  • Design experiments that both demonstrate physical interaction and functional consequences

How can non-specific binding be minimized when using TTC39B antibodies?

To minimize non-specific binding with TTC39B antibodies:

• Optimization of blocking reagents:

  • Test alternative blocking agents (BSA, casein, commercial blocking buffers)

  • For Western blots, 5% non-fat dry milk in TBST is often effective

  • For immunohistochemistry, normal serum from the secondary antibody host species (5-10%) can reduce background

• Antibody dilution optimization:

  • Perform titration experiments (typical ranges: 1:500-1:1000 for WB, 1:50-1:500 for IHC)

  • Higher dilutions may reduce non-specific binding while maintaining specific signal

• Cross-adsorption considerations:

  • Consider using antibodies that have been cross-adsorbed against related proteins

  • This is particularly important given the homology between TTC39B and its paralogs TTC39A and TTC39C

• Sample preparation refinements:

  • For tissues with high lipid content (like liver), additional washing steps may be necessary

  • Ensure complete removal of Laemmli buffer components before antibody incubation

• Secondary antibody selection:

  • Use highly cross-adsorbed secondary antibodies

  • Consider secondary antibodies specifically designed for the host species of your samples

How can discrepancies in observed molecular weights of TTC39B be interpreted?

When analyzing discrepancies in observed molecular weights of TTC39B:

• Expected versus observed weights:

  • Calculated molecular weight: 77 kDa (canonical form)

  • Commonly observed weights: 59 kDa and 67 kDa

• Potential explanations:

  • Isoform detection: Up to 7 different isoforms have been reported

  • Post-translational modifications: Phosphorylation, glycosylation, or ubiquitination may alter migration

  • Proteolytic processing: Functional fragments may be detected

  • Technical factors: SDS-PAGE conditions can affect migration patterns

• Verification approaches:

  • Use multiple antibodies targeting different epitopes to confirm identity

  • Perform mass spectrometry analysis of excised bands

  • Compare migration patterns in different tissues/cell types known to express specific isoforms

  • Consider knockdown/knockout samples as negative controls

• Functional implications:

  • Document which molecular weight forms correlate with specific functions

  • Different weights may represent functionally distinct pools of the protein

What strategies can resolve contradictory findings when using different TTC39B antibodies?

To resolve contradictory findings with different TTC39B antibodies:

• Comprehensive antibody validation:

  • Verify epitope locations for each antibody

  • Test all antibodies on the same positive and negative control samples

  • Compare results with mRNA expression data

• Epitope accessibility analysis:

  • Different antibodies may have varying access to epitopes depending on:

    • Protein conformation

    • Protein-protein interactions

    • Post-translational modifications

    • Fixation methods (for IHC/IF)

• Protocol standardization:

  • Ensure identical experimental conditions when comparing antibodies

  • Document differences in sample preparation, incubation times, and detection methods

• Isoform-specific detection:

  • Determine if discrepancies reflect different isoform detection patterns

  • Design experiments to specifically identify which isoforms each antibody recognizes

• Functional validation:

  • Use functional assays (e.g., LXR degradation, HDL metabolism) to determine which antibody results correlate with expected physiological outcomes

  • Combine protein detection with functional readouts to resolve contradictions

How can TTC39B antibodies facilitate research into novel therapeutic approaches for metabolic disorders?

TTC39B antibodies can advance therapeutic research for metabolic disorders through:

• Target validation studies:

  • Use antibodies to confirm TTC39B expression in patient samples

  • Correlate protein levels with disease severity metrics

  • Research has linked TTC39B deficiency to reduced mortality in high-fat/high-cholesterol diet models

• Compound screening approaches:

  • Develop cellular assays using TTC39B antibodies to identify compounds that modulate protein expression or function

  • Monitor changes in TTC39B-LXR interactions following treatment with candidate compounds

• Mechanistic investigations:

  • TTC39B deficiency has been shown to stabilize LXR, reducing both atherosclerosis and fatty liver disease

  • Use antibodies to track pathway components when testing potential interventions

• Biomarker development:

  • Evaluate TTC39B as a potential biomarker for metabolic disease progression

  • Correlate expression with established biomarkers like ALT (studies showed decreased ALT levels in TTC39B-deficient mice)

• Genetic therapy monitoring:

  • Use antibodies to assess protein levels following gene therapy approaches

  • Track compensatory changes in related pathways

What are the considerations for using TTC39B antibodies in multiplex immunoassays?

For incorporating TTC39B antibodies in multiplex immunoassays:

• Antibody compatibility assessment:

  • Test for cross-reactivity between TTC39B antibodies and other target antibodies

  • Verify that detection systems don't interfere with each other

• Optimization of multiplexed protocols:

  • Adjust antibody concentrations for balanced detection of all targets

  • May require compromise conditions that provide acceptable results for all analytes

• Panel design strategies:

  • Include TTC39B with functionally related proteins:

    • LXR isoforms (NR1H2 and NR1H3)

    • Metabolic regulators (SREBP, ABCA1, SCD1)

    • Inflammatory markers for comprehensive disease profiling

• Signal normalization approaches:

  • Include housekeeping proteins appropriate for the tissue/condition

  • Consider ratiometric analysis (e.g., TTC39B:LXR ratio) for functional insights

• Validation requirements:

  • Single-plex validation prior to multiplex implementation

  • Spike-in controls to verify detection specificity in the multiplexed environment

How might TTC39B antibodies contribute to understanding the protein's role beyond lipid metabolism?

To explore TTC39B functions beyond established lipid metabolism roles:

• Protein interactome mapping:

  • Use TTC39B antibodies for immunoprecipitation coupled with mass spectrometry

  • Identify novel interaction partners beyond the known LXR association

  • Given its TPR motifs, TTC39B likely functions as a scaffolding protein mediating multiple protein-protein interactions

• Subcellular localization studies:

  • Employ immunofluorescence with TTC39B antibodies to track dynamic localization

  • Changes in localization may indicate functions beyond lipid metabolism

  • Consider co-localization with organelle markers to identify potential novel functions

• Tissue-specific function analysis:

  • While liver and intestinal functions are established, explore expression and function in other tissues

  • The protein's high conservation across species suggests potentially undiscovered roles

• Developmental biology applications:

  • Investigate expression patterns during embryonic development

  • TPR-containing proteins often have roles in developmental processes

• Stress response investigations:

  • Monitor TTC39B expression and localization under various cellular stresses

  • TPR domain proteins frequently function in cellular stress responses and protein quality control