TRIAP1 Antibody

What is TRIAP1 and what cellular functions does it regulate?

TRIAP1, also known as p53CSV (p53-inducible cell survival factor), is a small 76-amino acid protein that functions as a prosurvival factor. It is the homolog of yeast Mdm35, a chaperone that interacts with Ups/PRELI family proteins and participates in intramitochondrial transfer of lipids for the synthesis of cardiolipin (CL) and phosphatidylethanolamine . TRIAP1 forms complexes with PRELI family members (PRELID1, PRELID3A, and PRELID3B) and carries out lipid transfer activities for phosphatidic acid (PA) and phosphatidylserine (PS) . Beyond its mitochondrial functions, TRIAP1 binds HSP70 in the cytoplasm to block formation of the apoptosome and caspase-9 activation, thus inhibiting apoptosis .

How is TRIAP1 expression regulated in normal versus cancer tissues?

TRIAP1 expression is ubiquitous across human tissues but shows significant variation in expression levels. Normal breast and non-tumorigenic breast cells exhibit lower TRIAP1 mRNA levels compared to breast cancer cells or their drug-resistant derivatives . In nasopharyngeal carcinoma (NPC), TRIAP1 is aberrantly overexpressed and associated with poor patient survival . At the molecular level, TRIAP1 expression is negatively regulated by microRNAs, particularly miR-320b, which directly targets the TRIAP1 3'UTR . Additionally, TRIAP1 expression can be induced by stress conditions, such as estrogen deprivation in MCF-7 breast cancer cells .

What criteria should be considered when selecting a TRIAP1 antibody for research applications?

When selecting a TRIAP1 antibody, researchers should consider:

• Application compatibility: Ensure the antibody has been validated for your intended application (WB, IHC, ELISA)

• Species reactivity: Confirm reactivity with your experimental model (human, mouse, rat, etc.)

• Antibody type: Consider whether a polyclonal or monoclonal antibody is more appropriate for your needs

• Epitope targeting: Understand which region of TRIAP1 the antibody recognizes

• Validation evidence: Review published literature using the antibody

For example, the Proteintech TRIAP1 antibody (15351-1-AP) has been validated for WB, IHC, and ELISA applications with demonstrated reactivity in human, mouse, and rat samples . It's a rabbit polyclonal antibody generated against a TRIAP1 fusion protein .

What controls are essential when validating a TRIAP1 antibody for experimental use?

Proper validation requires:

• Positive control: Lysates from cells known to express TRIAP1 (e.g., cancer cell lines like SUNE-1 or CNE-2)

• Negative control: Lysates from TRIAP1 knockdown cells using validated siRNAs (e.g., siTRIAP1-1: 5'-AGGCAUGCACGGACAUGAATT-3'; siTRIAP1-2: 5'-GAAAGAGAUUCCUAUUGAATT-3')

• Overexpression control: Lysates from cells transfected with TRIAP1 expression plasmids

• Loading control: Appropriate housekeeping protein (e.g., α-tubulin)

• Specificity test: Competition assay with the immunogen peptide

For IHC applications, additional controls should include tissues known to express TRIAP1 (e.g., human breast cancer tissue) and isotype control antibodies to assess non-specific binding .

What are the optimal protocols for TRIAP1 detection by Western blotting?

Based on published methodologies, the following protocol is recommended:

• Sample preparation: Lyse cells at 4°C using RIPA buffer containing protease inhibitor cocktail

• Protein separation: Use 12% SDS-PAGE gels (TRIAP1 is a small 9 kDa protein)

• Transfer: Transfer to PVDF membranes (Millipore or equivalent)

• Blocking: Block membranes with 5% non-fat milk in TBST

• Primary antibody: Incubate with anti-TRIAP1 antibody (e.g., 1:200 dilution for Santa Cruz antibody)

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody (e.g., 1:5000 dilution)

• Detection: Visualize using enhanced chemiluminescence

• Quantification: Normalize to loading controls such as α-tubulin (1:1000 dilution)

Note that different antibodies may require optimization of dilution ratios and incubation conditions.

How should immunohistochemistry procedures be optimized for TRIAP1 detection in tissue samples?

For optimal IHC detection of TRIAP1:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections

• Antigen retrieval: Use TE buffer pH 9.0 as the primary recommendation, with citrate buffer pH 6.0 as an alternative

• Antibody dilution: Start with a range of 1:50-1:500 for TRIAP1 antibody (15351-1-AP) and optimize based on signal intensity and background

• Controls: Include positive control tissues such as mouse pancreas tissue or human breast cancer tissue

• Detection system: Use an appropriate secondary antibody and visualization system compatible with your primary antibody

• Counterstaining: Use hematoxylin for nuclear counterstaining

The specific protocol may need optimization based on the tissue type and fixation methods used.

How can TRIAP1 knockdown or overexpression be achieved for functional studies?

Several validated approaches have been documented:

For TRIAP1 knockdown:

• siRNA-mediated knockdown:

  • siTRIAP1-1: 5'-AGGCAUGCACGGACAUGAATT-3'

  • siTRIAP1-2: 5'-GAAAGAGAUUCCUAUUGAATT-3'

  • Transfect using Lipofectamine 2000 at 100 nM concentration

• shRNA-mediated stable knockdown:

  • Clone synthesized shTRIAP1 into pSuper-retro-puromycin vectors

  • Generate stable cell lines through lentiviral infection

  • Select with 0.5 μg/ml puromycin

For TRIAP1 overexpression:

• Transient overexpression:

  • Clone human TRIAP1 gene into expression vectors

  • Transfect using Lipofectamine 2000 (2 μg plasmid)

• Stable overexpression:

  • Clone TRIAP1 into the pSin-EF2-puromycin vector

  • Generate stable cell lines through lentiviral infection

  • Select with puromycin

Functional validation of knockdown or overexpression should be performed at both mRNA and protein levels before proceeding with functional assays.

What cellular assays are most informative for studying TRIAP1 function in cancer cells?

Based on TRIAP1's documented functions, these assays provide valuable insights:

• Proliferation assays: MTT, colony formation, or EdU incorporation assays to assess effects on cell growth

• Apoptosis assays:

  • Annexin V/PI staining for flow cytometry

  • TUNEL assay for in situ detection of apoptotic cells

  • Western blot for apoptotic markers (cleaved caspase-3, cytochrome c)

• Mitochondrial function assays:

  • Mitochondrial membrane potential using JC-1 or TMRM dyes

  • Mitochondrial morphology by fluorescence microscopy

  • Analysis of cytochrome c release from mitochondria to cytosol

• Drug resistance assays:

  • Cell viability assays in the presence of chemotherapeutic agents (e.g., doxorubicin)

  • Combination treatment with glutamine deprivation

• In vivo tumor growth:

  • Subcutaneous xenograft models using TRIAP1-overexpressing or knockdown cells

  • Tumor volume and weight measurements

  • IHC analysis of proliferation markers (Ki67) and TUNEL assay for apoptosis

How does TRIAP1 contribute to cancer cell proliferation and survival?

TRIAP1 promotes cancer development through multiple mechanisms:

• Apoptosis inhibition: TRIAP1 binds HSP70 in the cytoplasm and blocks formation of the apoptosome and caspase-9 activation . In NPC cells, TRIAP1 knockdown enhances apoptosis through the induction of mitochondrial fragmentation, membrane potential alteration, and release of cytochrome c from mitochondria into the cytosol .

• Proliferation enhancement: TRIAP1 overexpression promotes cancer cell proliferation in vitro and in vivo, while its knockdown inhibits tumorigenesis .

• Drug resistance: TRIAP1 is upregulated in drug-resistant cancer cells. Overexpression of TRIAP1 increases resistance to doxorubicin in breast cancer cells, while its knockdown enhances drug sensitivity .

• Metabolic adaptation: TRIAP1 depletion causes changes in endoplasmic reticulum-dependent lipid homeostasis. Interestingly, TRIAP1 depletion confers robust p53-mediated resistance to metabolic stress caused by glutamine deprivation .

• Mitochondrial function: TRIAP1 partners with PRELID proteins to facilitate intramitochondrial lipid transfer, which is essential for maintaining mitochondrial integrity and function .

What is the relationship between TRIAP1, p53 signaling, and mitochondrial function?

TRIAP1 has complex interactions with p53 signaling and mitochondrial function:

How do microRNAs regulate TRIAP1 expression and what are the implications for cancer biology?

MicroRNA regulation of TRIAP1 has significant implications for cancer development:

• miR-320b as a direct regulator: miR-320b directly targets TRIAP1 by binding to its 3'UTR at both conserved (position 265-272) and poorly conserved (position 550-556) binding sites .

• Expression patterns: miR-320b is downregulated in nasopharyngeal carcinoma tissues, which inversely correlates with TRIAP1 overexpression .

• Functional consequences:

  • miR-320b overexpression suppresses NPC cell proliferation and enhances mitochondrial fragmentation and apoptosis both in vitro and in vivo

  • Silencing of miR-320b promotes tumor growth and suppresses apoptosis

  • TRIAP1 restoration abrogates the proliferation inhibition and apoptosis induced by miR-320b

• Experimental validation: Luciferase reporter assays confirm that miR-320b directly targets the TRIAP1 3'UTR, with miR-320b mimics significantly inhibiting TRIAP1 expression at both mRNA and protein levels .

• Therapeutic potential: The miR-320b/TRIAP1 axis represents a potential therapeutic target, where miR-320b mimics might be used to suppress TRIAP1 expression and inhibit cancer growth .

What are common technical challenges when detecting TRIAP1 by Western blot and how can they be addressed?

Detection of TRIAP1 (9 kDa) by Western blot can be challenging due to:

• Small protein size: Use higher percentage gels (12-15% SDS-PAGE) and adjust transfer conditions for small proteins (higher current, shorter time, or specialized transfer buffers with methanol) .

• Low expression levels: Increase protein loading (50-100 μg per lane) and optimize primary antibody concentration.

• Antibody sensitivity issues: Historical challenges with antibody detection have been noted, as "both polyclonal and monoclonal antibodies generated for the present study failed to robustly detect TRIAP1" . Try commercial antibodies with validated applications in Western blot.

• Non-specific binding: Increase blocking time/concentration, use TBST with higher Tween-20 concentration (0.1-0.2%), and optimize antibody dilutions.

• Sample preparation: Ensure complete protein extraction using appropriate lysis buffers with protease inhibitors at 4°C as described in published protocols .

• Detection method: Use highly sensitive ECL substrates or consider fluorescent secondary antibodies for better quantification.

How can discrepancies in TRIAP1 expression data between different detection methods be reconciled?

When facing contradictory results across different detection methods:

• Validate antibody specificity:

  • Use positive controls (cancer cell lines known to express TRIAP1)

  • Include TRIAP1 knockdown and overexpression controls

  • Consider using multiple antibodies targeting different epitopes

• Cross-validate at mRNA level:

  • Perform RT-qPCR to assess TRIAP1 mRNA levels

  • Compare with protein expression data to identify post-transcriptional regulation

• Consider technical differences:

  • WB detects denatured protein, while IHC detects proteins in their native conformation/location

  • Different fixation methods in IHC can affect epitope accessibility

  • WB provides average expression across cell populations, while IHC shows cellular localization

• Optimize detection methods:

  • For WB: Try different extraction methods, gel percentages, and transfer conditions

  • For IHC: Test different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Adjust antibody concentrations based on application (1:50-1:500 for IHC)

• Document experimental conditions thoroughly:

  • Cell/tissue type, sample preparation method, antibody used

  • For reproducibility and accurate comparison between experiments