TINAGL1 Antibody

What is TINAGL1 and why is it significant in research?

TINAGL1 (Tubulointerstitial nephritis antigen-like 1) is a 52-57 kDa secreted glycoprotein member of the peptidase C1 family. Initially identified as a putative component of the extracellular matrix (ECM), TINAGL1 plays significant roles in:

• Embryonic development, particularly of the heart and adrenal glands

• Cell adhesion, migration, and invasion processes

• Cancer biology, especially in hepatocellular carcinoma and breast cancer

TINAGL1 contains multiple structural domains including an SMB (somatomedin B) domain (aa 50-98), a vWFC domain (aa 105-140), and a nonenzymatic peptidase C1A region (aa 204-455) . Its significance in research stems from its emerging roles in both developmental processes and pathological conditions, making TINAGL1 antibodies valuable tools for investigating these biological contexts .

What applications are TINAGL1 antibodies validated for?

TINAGL1 antibodies have been validated for multiple research applications with specific optimization parameters:

For optimal results, each antibody should be titrated for specific experimental conditions. Sample-dependent variables often require protocol optimization . For Western blot detection, both reducing conditions and specific buffer systems (such as Immunoblot Buffer Group 1) have proven effective for visualizing the expected 52-59 kDa band .

How should TINAGL1 antibodies be stored and handled for optimal performance?

Proper storage and handling of TINAGL1 antibodies are crucial for maintaining reactivity and specificity:

• Storage temperature: Most TINAGL1 antibodies should be stored at -20°C for standard polyclonal antibodies or -80°C for more sensitive preparations

• Buffer composition: Typically supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Stability: Generally stable for one year after shipment when properly stored

• Aliquoting: While some formulations state aliquoting is unnecessary for -20°C storage, dividing into single-use aliquots is recommended for antibodies requiring frequent use to avoid freeze-thaw cycles

• Reconstitution (if lyophilized): Follow manufacturer guidelines precisely, as improper reconstitution can reduce antibody performance

For long-term experimental planning, consider that properly stored antibodies maintain reactivity for detection of TINAGL1 in human, mouse, and rat samples across multiple applications .

What is the expected molecular weight of TINAGL1 and how might it vary?

The molecular weight of TINAGL1 varies slightly depending on detection methods and tissue sources:

This variation is attributed to:

• Post-translational modifications, primarily glycosylation

• Tissue-specific processing

• Presence of multiple isoform variants

Research has identified several potential isoform variants, including one with an alternative start site at Met106, another showing a deletion of aa 126-156, and a third with a 60 aa substitution for aa 1-194 coupled to additional modifications . Additionally, undefined forms of 40-42 kDa and 22 kDa have been observed in TINAGL1-transfected cell supernatants subjected to SDS-PAGE, suggesting further proteolytic processing or alternative splicing events .

How can I optimize immunohistochemical detection of TINAGL1?

For optimal immunohistochemical detection of TINAGL1 in tissue samples:

Antigen Retrieval Methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Protocol Optimization:

• Section preparation: 4-5 μm sections from formalin-fixed, paraffin-embedded tissues provide optimal results

• Blocking: 5% normal serum from the same species as the secondary antibody for 1 hour at room temperature

• Primary antibody incubation: Use validated TINAGL1 antibodies at 1:50-1:500 dilution; overnight incubation at 4°C improves signal-to-noise ratio

• Detection system: Polymer-based detection systems typically produce cleaner results than avidin-biotin methods

• Counterstaining: Light hematoxylin counterstaining allows better visualization of TINAGL1-positive structures

TINAGL1 protein has been successfully detected in the cytoplasm of cancer cells, particularly in liver cancer tissue samples . For breast cancer studies, IHC has been effectively used to localize TINAGL1 in the cytoplasm of cancer cells using antibodies like HPA048695 .

How can TINAGL1 antibodies be used to investigate its role in cancer progression?

TINAGL1 antibodies serve as critical tools for investigating the complex roles of this protein in cancer biology through multiple experimental approaches:

In Hepatocellular Carcinoma (HCC):
TINAGL1 promotes carcinogenesis and metastasis via the TGF-β/Smad3/VEGF axis. Researchers have successfully employed the following methodologies:

• Expression analysis: Western blotting with TINAGL1 antibodies (1:1,000 dilution) demonstrated significantly higher TINAGL1 levels in HCC tissues compared to adjacent non-tumor tissues

• Signaling pathway investigation: Combined immunoblotting for TINAGL1, Smad3, and VEGF enabled researchers to elucidate that:

  • TGF-β stimulation (5 ng/mL) activated Smad3 and increased VEGF secretion

  • TINAGL1 upregulated both Smad3 and VEGF

  • Smad3 inhibition by SB431542 suppressed VEGF secretion

• Functional studies: Knockdown and overexpression experiments followed by proliferation, migration, and apoptosis assays demonstrated that TINAGL1 silencing inhibited proliferation, migration, and invasion while inducing apoptosis

In Breast Cancer:
Contrasting with HCC, research has shown that TINAGL1 may suppress tumor metastasis and growth in triple-negative breast cancer:

• Prognostic correlation: IHC analysis of 299 breast cancer patient samples and RT-PCR analysis of 599 samples revealed that low TINAGL1 mRNA expression correlates with worse prognosis and shorter disease-free survival

• Expression discrepancy analysis: The lack of correlation between TINAGL1 protein and mRNA expression highlights the importance of post-transcriptional regulation mechanisms that can be investigated using both antibody-based protein detection and mRNA quantification methods

This dual role of TINAGL1 as both pro-tumorigenic and tumor-suppressive in different cancer contexts demonstrates the importance of tissue-specific investigation using validated antibodies.

What methodological approaches can resolve discrepancies between TINAGL1 mRNA and protein expression?

The discrepancy between TINAGL1 mRNA and protein expression observed in breast cancer research presents a complex challenge requiring sophisticated methodological approaches:

Integrated Analysis Approaches:

• Polysome profiling with immunoblotting:

  • Fractionate polysomes from tissue samples using sucrose gradient centrifugation

  • Extract RNA from fractions for TINAGL1 mRNA quantification by RT-PCR

  • Simultaneously assess protein levels by Western blot using validated TINAGL1 antibodies

  • This approach reveals whether translational efficiency contributes to the discrepancy

• Protein stability assessment:

  • Treat cells with protein synthesis inhibitors (e.g., cycloheximide)

  • Use TINAGL1 antibodies to monitor protein degradation rates via Western blot at various timepoints

  • Compare degradation kinetics between different cancer subtypes or tissue contexts

• Post-translational modification analysis:

  • Immunoprecipitate TINAGL1 using specific antibodies

  • Analyze precipitates by mass spectrometry to identify modifications

  • Compare modification patterns between samples with divergent mRNA-protein correlations

Technical considerations for accurate comparison:

• Use matched samples for both mRNA and protein analysis

• Implement absolute quantification methods for both modalities

• Validate antibody specificity using knockdown/knockout controls

• Consider subcellular localization effects by fractionating samples prior to analysis

The observation that TINAGL1 protein expression lacks correlation with mRNA expression, particularly in breast cancer studies , highlights the importance of post-transcriptional and post-translational regulatory mechanisms that can significantly impact biomarker development and interpretation.

How do monoclonal and polyclonal TINAGL1 antibodies compare in research applications?

Monoclonal and polyclonal TINAGL1 antibodies offer distinct advantages and limitations that should inform selection based on specific research applications:

Monoclonal TINAGL1 Antibodies:

The Mouse Anti-Human TINAGL1 Monoclonal Antibody (e.g., MAB7185, Clone #812417) offers:

• Epitope specificity: Recognizes a specific region (Ala22-Met464) of human TINAGL1

• Consistent lot-to-lot reproducibility

• Highly specific detection in Western blot (~55 kDa) and Simple Western (~59 kDa) applications

• Validated for human placenta and kidney tissue lysates under reducing conditions

• Potentially limited in detecting certain isoforms or heavily modified variants

Polyclonal TINAGL1 Antibodies:

Rabbit Polyclonal TINAGL1 Antibodies (e.g., 12077-1-AP, CAB13122) provide:

• Recognition of multiple epitopes across the TINAGL1 protein

• Broader reactivity across species (human, mouse, rat)

• Versatility across multiple applications (WB, IHC, IF/ICC, IF-P, IP, ELISA)

• Enhanced sensitivity for detecting native conformations

• Variable lot-to-lot consistency requiring validation

Comparative Performance Analysis:

For critical research applications, validation experiments comparing both antibody types on the same samples can determine which antibody type provides optimal results for specific experimental conditions and biological questions.

How can TINAGL1 antibodies be used to investigate the TGF-β/Smad3/VEGF signaling axis?

TINAGL1 antibodies provide powerful tools for dissecting the TGF-β/Smad3/VEGF signaling axis, particularly in hepatocellular carcinoma where this pathway drives tumor progression:

Experimental Approaches:

• Co-immunoprecipitation of signaling complexes:

  • Immunoprecipitate TINAGL1 using validated antibodies

  • Probe precipitates for TGF-β pathway components (TGF-β receptors, Smad proteins)

  • Identify novel protein interactions that may mediate TINAGL1's effects on signaling

• Chromatin immunoprecipitation (ChIP) analysis:

  • Use antibodies against Smad3 to immunoprecipitate chromatin

  • Perform qPCR or sequencing on precipitated DNA to identify VEGF promoter regions

  • Compare ChIP efficiency between TINAGL1-overexpressing and control cells

• Proximity ligation assay (PLA):

  • Utilize TINAGL1 antibodies in combination with antibodies against TGF-β pathway components

  • Visualize and quantify protein-protein interactions in situ

  • Determine spatial relationships between TINAGL1 and signaling components

Methodological Protocol for Signaling Analysis:

• Sample preparation:

  • Culture HCC cells under defined conditions (with/without TGF-β stimulation)

  • Consider TINAGL1 overexpression or knockdown conditions

  • Extract proteins using buffers that preserve phosphorylation states

• Western blot analysis:

  • Use TINAGL1 antibodies (1:1,000 dilution) alongside antibodies against:

    • Smad3 (1:1,000, #25494-1-AP)

    • VEGF (1:1,000, #19003-1-AP)

    • Phosphorylated Smad3 (to detect activation)

  • Include GAPDH (1:1,000, #10494-1-AP) as loading control

• Functional validation:

  • Combine antibody-based detection with inhibitor studies

  • Use TGF-β pathway inhibitors (e.g., SB431542) to confirm signaling relationships

  • Quantify VEGF secretion using ELISA following pathway manipulation

Research has demonstrated that TINAGL1 promotes hepatocellular carcinogenesis by activating the TGF-β signaling pathway and increasing VEGF secretion, making this axis a critical target for mechanistic studies and potential therapeutic intervention .

What validation techniques ensure reliable results with TINAGL1 antibodies?

Comprehensive validation of TINAGL1 antibodies is essential for generating reliable, reproducible research data:

Critical Validation Approaches:

• Genetic validation using knockdown/knockout systems:

  • Generate TINAGL1 knockdown cells using siRNA (as demonstrated in HCC studies)

  • Create CRISPR/Cas9 knockout cell lines or utilize TINAGL1-/- mouse models

  • Confirm antibody specificity by demonstrating signal reduction/elimination

• Western blot validation parameters:

  • Positive controls: Human placenta and kidney tissues show consistent TINAGL1 expression

  • Expected molecular weight verification: 52-59 kDa depending on tissue source and detection method

  • Multiple antibody comparison: Test monoclonal and polyclonal antibodies against the same samples

  • Blocking peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

• Immunohistochemistry validation:

  • Tissue processing controls: Standardize fixation and antigen retrieval methods

  • Antibody titration: Test dilutions from 1:50 to 1:500 to determine optimal signal-to-noise ratio

  • Positive tissue controls: Human liver cancer tissue has demonstrated reliable TINAGL1 detection

  • Negative controls: Include secondary-only controls and non-expressing tissues

• Application-specific validation:

Implementing these validation strategies ensures that experimental observations truly reflect TINAGL1 biology rather than technical artifacts, particularly critical when investigating complex cancer mechanisms or developing TINAGL1 as a potential biomarker.