TRAP1 Antibody

What epitopes are most commonly targeted by commercial TRAP1 antibodies?

Commercial TRAP1 antibodies target different regions of the protein depending on their intended applications. Some antibodies like ab226401 target synthetic peptides within the 600-650 amino acid region of human TRAP1 , while others such as MA1-010 use purified, recombinant, full-length human TRAP1 as the immunogen . When selecting a TRAP1 antibody, consider whether the epitope is located in a conserved region (important for cross-species reactivity) and whether it might be masked in certain experimental conditions due to protein folding or interactions with other proteins.

The choice of epitope significantly impacts the antibody's performance across different applications and its cross-reactivity with TRAP1 from different species. For studying specific post-translational modifications or particular functional domains of TRAP1, epitope location becomes especially critical.

What are the key differences between monoclonal and polyclonal TRAP1 antibodies for research applications?

Monoclonal TRAP1 antibodies:

• Recognize a single epitope on the TRAP1 protein

• Provide high specificity but potentially lower sensitivity

• Produce consistent results between batches

• Examples include ab2721 (clone TRAP1-6) and MA1-010

Polyclonal TRAP1 antibodies:

• Recognize multiple epitopes on the TRAP1 protein

• Offer higher sensitivity but with potential increased background

• May show batch-to-batch variation

• Examples include ab226401

The choice depends on your research application. Monoclonal antibodies are preferred for applications requiring high specificity, while polyclonal antibodies may be advantageous for detecting low-abundance TRAP1 or for applications like immunoprecipitation where recognition of multiple epitopes improves protein capture.

What are the optimal conditions for Western blot analysis using TRAP1 antibodies?

Based on published research, the following conditions optimize TRAP1 detection by Western blot:

Sample preparation:

• Use NETN lysis buffer for lysate preparation

• Load 30 µg of whole cell lysate per lane (HeLa, HepG2, HEK-293, or K-562 cells work well as positive controls)

Antibody dilutions:

• Primary: ab2721 at 1/2000 or ab226401 at 0.04 µg/mL

• Secondary: Anti-mouse IgG-HRP at 1/40000 for ab2721

Detection parameters:

• Expected molecular weight: 75-80 kDa

• Note that TRAP1 often appears at approximately 75 kDa despite a predicted size of 80 kDa , possibly due to post-translational modifications

Controls:

• Positive controls: HeLa or HepG2 cell lysates

• Negative controls: Lysates from TRAP1 knockout or knockdown cells

For validation, researchers should note that TRAP1 deficiency promotes increased mitochondrial respiration and ATP levels, which can be measured as functional readouts . TRAP1 silencing efficacy should always be verified by parallel Western blot analysis before proceeding to functional studies .

How can TRAP1 antibodies be effectively used to investigate subcellular localization?

TRAP1 was originally characterized as predominantly mitochondrial, but recent studies have revealed its presence in the endoplasmic reticulum as well . This dual localization makes proper immunofluorescence techniques essential:

Protocol for immunofluorescence:

• Fix cells with 4% paraformaldehyde (10 minutes)

• Permeabilize and block with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween (1 hour)

• Incubate with primary TRAP1 antibody (e.g., ab2721 at 5µg/ml) overnight at 4°C

• Apply appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488)

• Co-stain with compartment markers:

  • Mitochondria: MitoTracker or antibodies against TOM20

  • ER: Antibodies against calnexin or PDI

• Counterstain nuclei with DAPI (1.43µM concentration)

• Image using confocal microscopy

For distinguishing between mitochondrial and ER pools of TRAP1, subcellular fractionation followed by Western blot can provide quantitative data to complement imaging approaches. The discovery that "TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses" has important implications for understanding TRAP1's diverse cellular functions.

What controls are essential when using TRAP1 antibodies for immunoprecipitation experiments?

Immunoprecipitation with TRAP1 antibodies requires rigorous controls to ensure specificity and reliability:

Essential controls:

• No-antibody control: Include a sample processed identically but without adding TRAP1 antibody

• Isotype control: Use an irrelevant antibody of the same isotype as the TRAP1 antibody

• Input sample: Run a portion (5-10%) of the starting material alongside IP samples

• TRAP1 knockout/knockdown control: If available, include lysate from cells with verified TRAP1 depletion

Recommended protocol:

• Incubate 5μg of TRAP1 antibody with 50μl of protein G magnetic beads for 10 minutes under agitation

• Add 0.5mg of cell extract (e.g., HepG2) and incubate for 10 minutes under agitation

• Elute proteins with SDS loading buffer (70°C for 10 minutes)

• Analyze by SDS-PAGE and Western blot

Importantly, immunoprecipitation studies have revealed that TRAP1 does not associate with co-chaperones like p23, Hop, or CyP40 , distinguishing it from other HSP90 family members. More recent research has identified important TRAP1 interactions, including with F-ATP synthase, where "TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase" .

How can TRAP1 antibodies be used to investigate metabolic reprogramming in cancer cells?

TRAP1's role in regulating the balance between oxidative phosphorylation and glycolysis makes it a key target for investigating metabolic reprogramming in cancer. Several experimental approaches using TRAP1 antibodies can elucidate these mechanisms:

Correlation studies:

• Quantify TRAP1 protein levels in tissue samples or cell lines using Western blot or immunohistochemistry with validated antibodies

• Measure metabolic parameters in the same samples:

  • Oxygen consumption rate (OCR)

  • Extracellular acidification rate (ECAR)

  • ATP levels

  • Reactive oxygen species (ROS)

• Perform statistical analysis to identify correlations

Research has demonstrated that "TRAP1 deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species" . This supports TRAP1's role as a negative regulator of oxidative phosphorylation.

Gain/loss-of-function approaches:

• Verify TRAP1 manipulation (overexpression, silencing, knockout) using Western blot

• Measure changes in metabolic parameters:

  • TRAP1 knockdown: ~2-fold increase in OCR in isolated mitochondria

  • TRAP1 overexpression: Significant reduction in OCR and cellular ATP levels

Studies in colorectal cancer have revealed a negative correlation between TRAP1 expression and mitochondrial gene expression, where "TRAP1-silencing induced a significant increase in all 13 mt-genes expression compared to control" , providing further evidence of TRAP1's metabolic regulatory function.

How can researchers study TRAP1's interaction with F-ATP synthase using antibody-based methods?

The interaction between TRAP1 and F-ATP synthase has significant implications for cellular bioenergetics and survival. Several antibody-based approaches can investigate this interaction:

Co-immunoprecipitation approaches:

• Immunoprecipitate TRAP1 using validated antibodies, then probe for F-ATP synthase subunits by Western blot

• Perform reverse co-IP using antibodies against OSCP (the F-ATP synthase subunit that interacts with TRAP1)

• Include appropriate controls as outlined in section 2.3

Competitive binding analysis:

• Design experiments to test competition between TRAP1 and CyPD for binding to OSCP

• Manipulate CyPD levels and assess TRAP1-OSCP interaction by co-IP

• Analyze the effect on F-ATP synthase activity and channel formation

Research has shown that "TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death" . This demonstrates TRAP1's role in regulating mitochondrial function beyond metabolic control.

Split GFP complementation:
This technique allows visualization of protein interactions in living cells. sMPNST TRAP1 knockout cells can be transfected with a combination of plasmids including pcDNA3 mito-GFP1-9, pcDNA3-TRAP1-GFP10, and pcDNA3ATP50-GFP11 . When TRAP1 and ATP50 interact, the GFP fragments complement each other, producing fluorescence that can be detected microscopically.

What methodologies enable investigation of TRAP1's relationship with client proteins in cancer?

TRAP1 interacts with numerous client proteins, forming a network that influences cancer progression and therapeutic response. Antibody-based methods to study these relationships include:

Proteomic signature analysis:

• Quantify TRAP1 and client proteins (F1ATPase, TBP7, IF2α, EF1G, IF4A, IF4E, EF1A, BRAF, AKT, Sorcin, CDK1, MAD2, βCatenin) by immunoblotting in cancer samples

• Perform correlation analysis using Spearman Rank test

• Generate cluster analyses to identify patient subgroups

Multiplex immunohistochemistry:
This technique allows simultaneous detection of TRAP1 and multiple client proteins in tissue sections, enabling spatial relationship analysis at the cellular level.

Co-expression analysis in clinical databases:

• Extract TRAP1 and client protein expression data from cancer genomics databases

• Perform correlation analyses and survival associations

• Validate findings using immunohistochemistry on tissue samples

Importantly, "TRAP1 expression was quantified by immunohistochemistry, yielding 80% of cases with TRAP1 upregulation" in metastatic colorectal cancer, and "tumors with high TRAP1 expression are characterized by a worst outcome compared to tumors with low TRAP1 expression (HR 2.7; 95% C.I. 1.0-7.3; p=0.044)" .

How should researchers interpret discrepancies between TRAP1 mRNA and protein levels in cancer samples?

Discrepancies between TRAP1 mRNA and protein levels are common and reflect complex regulatory mechanisms. When encountering such discrepancies, consider:

Post-transcriptional regulation:
While TRAP1 copy number correlates with mRNA expression, the correlation coefficients are modest (R=0.32 and R=0.16) , indicating that copy number only partially explains expression levels. Other regulatory mechanisms likely play important roles.

Post-translational modifications:
TRAP1 function is regulated by PTMs , which could affect protein stability without changing mRNA levels. These modifications may also impact antibody recognition, potentially leading to apparent discrepancies.

Protein stability differences:
TRAP1 protein may have different degradation rates across tissues or disease states, creating a mismatch between mRNA and protein levels.

Technical considerations:

• Ensure antibody specificity through proper validation

• Consider sensitivity differences between mRNA and protein detection methods

• Verify sample quality and processing consistency

Research in colorectal cancer has revealed an inverse relationship where "a significant upregulation of TRAP1 expression was observed in cancer tissues [...]. Conversely, the expression of 13 mt-genes (mt-signature) was significantly downregulated in malignant tissues" . This pattern demonstrates the complex regulatory relationship between TRAP1 and mitochondrial function.

What are the most common technical challenges when using TRAP1 antibodies for cancer tissue analysis?

Researchers face several technical challenges when analyzing TRAP1 in cancer tissues:

Heterogeneous expression:
TRAP1 is overexpressed in 60-70% of human colorectal cancers , but its expression varies significantly between cancer types. In small cell lung cancer, "the expression of TRAP1 was low in SCLC and NSCLC compared with other groups, and was the lowest in SCLC" . This heterogeneity requires careful sampling and interpretation.

Variable subcellular localization:
TRAP1 localizes to both mitochondria and ER , and the distribution between these compartments may vary by cell type or disease state. Different antibodies may preferentially detect one pool over the other.

Tissue processing effects:
Fixation methods significantly impact epitope preservation. For optimal results, "heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer and microwaved for 8-15 minutes" before immunostaining.

Quantification challenges:
Determining what constitutes "positive" or "high" TRAP1 expression requires standardized scoring systems. Studies report that "TRAP1 expression was quantified by immunohistochemistry, yielding 80% of cases with TRAP1 upregulation" , but specific quantification criteria should be clearly defined.

Context-dependent interpretation:
TRAP1's prognostic significance varies across cancer types. In colorectal cancer, high expression correlates with poor outcome , while in small cell lung cancer, low TRAP1 expression "was negatively correlated with the occurrence of the disease" . This context-dependence necessitates cancer-specific interpretation.

How can researchers optimize TRAP1 antibody performance for challenging applications?

When facing challenges with TRAP1 antibody performance, consider these optimization strategies:

For high background in immunohistochemistry:

• Optimize blocking (try 3% BSA-PBS for 30 minutes at room temperature)

• Titrate antibody concentration (ab2721 has been used at 1:20 dilution)

• Quench endogenous peroxidase activity thoroughly

• Consider switching from polyclonal to monoclonal antibodies for higher specificity

• Test different antigen retrieval methods

For weak signal detection:

• Try signal amplification systems (e.g., tyramide signal amplification)

• Optimize antigen retrieval (10mM sodium citrate pH6.0, microwave 8-15 minutes)

• Use more sensitive detection systems (e.g., Super Sensitive Polymer-HRP)

• For Western blot, increase protein loading or use enhanced chemiluminescence substrates

For co-localization studies:

• Use super-resolution microscopy techniques

• Apply spectral unmixing to resolve overlapping fluorophores

• Perform subcellular fractionation to complement imaging data

• Consider proximity ligation assays to confirm direct interaction between TRAP1 and potential partners

For reproducibility challenges:

• Standardize sample processing (fixation times, buffer compositions)

• Include multiple positive and negative controls in each experiment

• Validate key findings with at least two independent TRAP1 antibodies

• Complement antibody-based detection with functional assays

When investigating TRAP1 and its client proteins, researchers found that "Spearman Rank correlation test showed a statistically significant co-expression between TRAP1 and most of its client proteins" , highlighting the importance of examining TRAP1 in the context of its protein network rather than in isolation.

How can TRAP1 antibodies be used to explore the link between mitochondrial dysfunction and disease?

TRAP1 antibodies offer powerful tools for investigating mitochondrial dysfunction in various diseases:

Autoinflammatory conditions:
Research has identified "homozygous mutations in TRAP1, encoding the mitochondrial/ER resident chaperone protein" in patients with severe autoinflammation . TRAP1 antibodies can be used to study how defective TRAP1 contributes to cellular stress and elevated IL-18 levels observed in these patients.

Neurodegenerative diseases:
While not extensively covered in the search results, TRAP1's role in mitochondrial homeostasis suggests potential involvement in neurodegenerative conditions. Antibodies could be used to investigate TRAP1 expression and localization in neuronal models and patient samples.

Cancer metabolism:
TRAP1 antibodies can reveal metabolic vulnerabilities in tumors, as "TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis" . This knowledge could inform development of metabolism-targeting therapies.

Research has demonstrated that "Impaired TRAP1 function leads to cellular stress and elevated levels of serum IL-18" , connecting mitochondrial dysfunction with inflammatory pathways. TRAP1 antibodies provide essential tools for dissecting these molecular mechanisms.

What role do TRAP1 antibodies play in developing potential therapeutic approaches?

TRAP1 antibodies contribute to therapeutic development in several ways:

Target validation:
Antibodies help validate TRAP1 as a therapeutic target by confirming its expression in disease tissues and its association with clinical outcomes. In colorectal cancer, "TRAP1 protein network may provide a prognostic signature" , supporting its relevance as a target.

Patient stratification:
TRAP1 antibodies can identify patient subgroups likely to respond to specific therapies. Research has shown that "tumors with high TRAP1 expression are characterized by a worst outcome compared to tumors with low TRAP1 expression" in metastatic colorectal cancer.

Monitoring treatment response:
Changes in TRAP1 expression or localization following treatment could serve as pharmacodynamic markers, with antibodies enabling this monitoring.

Therapeutic antibody development:
While not directly discussed in the search results, understanding TRAP1 biology through antibody-based research could inform development of therapeutic antibodies or antibody-drug conjugates targeting TRAP1-expressing cells.

The search results indicate that "TRAP1 expression levels influence mitochondrial architecture of human neuroblastoma cells and tumour metastasis in vivo" , suggesting that targeting TRAP1 could affect not only metabolism but also metastatic potential.