TFF3 Antibody, HRP conjugated

What are the primary applications for TFF3 antibodies in experimental research?

TFF3 antibodies are valuable tools in several experimental techniques, with HRP-conjugated variants offering particular advantages for detection-based assays. The primary applications include:

• Western Blot (WB): Typically used at dilutions ranging from 1:300-5000, enabling detection of TFF3 protein at approximately 12-14 kDa under reducing conditions . Some studies have detected higher molecular weight forms (up to 68 kDa), which may represent post-translationally modified variants or oligomeric forms .

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-P) and frozen (IHC-F) specimens can be analyzed with recommended dilutions of 1:200-400 and 1:100-500, respectively .

• ELISA: For quantitative analysis of TFF3 in biological samples at dilutions of approximately 1:500-1000 .

The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal.

What is the typical reactivity profile of commercially available TFF3 antibodies?

The reactivity profile varies between antibodies, but most commercially available TFF3 antibodies demonstrate:

• Confirmed reactivity: Human TFF3 is recognized by the majority of available antibodies, with some also cross-reacting with mouse and rat TFF3 .

• Predicted reactivity: Some antibodies may cross-react with bovine TFF3 based on sequence homology, though experimental validation is often needed .

• Cross-reactivity within TFF family: High-quality antibodies typically show minimal cross-reactivity with other TFF family members (TFF1, TFF2), usually less than 5% in direct ELISAs , enabling specific detection of TFF3.

How should TFF3-HRP conjugated antibodies be stored to maintain optimal activity?

Proper storage is critical for antibody performance. Based on manufacturer recommendations:

• Storage temperature: Store at -20°C for long-term stability .

• Aliquoting: Divide into multiple vials to avoid repeated freeze-thaw cycles that can compromise antibody functionality .

• Buffer conditions: Most are supplied in aqueous buffered solutions containing TBS (pH 7.4) with 1% BSA, preservatives like 0.02% Proclin300, and 50% glycerol for stability .

• Shelf-life: Typically stable for 12 months from receipt when stored according to recommendations .

How can I validate the specificity of a TFF3 antibody in my experimental system?

Thorough validation is essential for reliable results. Recommended approaches include:

• Positive controls: Use tissues/cell lines known to express TFF3, such as human pancreatic tissue, KATO-III gastric carcinoma cells, or LNCaP prostate cancer cells .

• Blocking peptides: Test antibody specificity by pre-incubation with the immunogenic peptide used to generate the antibody, which should abolish specific staining .

• siRNA knockdown: Confirm specificity by analyzing samples with TFF3 expression silenced via siRNA, which should show significantly reduced signal compared to controls .

• Western blot analysis: Verify the expected molecular weight (approximately 12-14 kDa for monomeric TFF3) under reducing conditions .

What are the optimal conditions for detecting TFF3 in Western blot applications?

For optimal Western blot detection of TFF3:

• Sample preparation: Use RIPA buffer with EDTA and protease inhibitors for effective protein extraction .

• Electrophoresis conditions: 12-15% SDS-polyacrylamide gels are recommended for optimal resolution of the relatively small TFF3 protein .

• Antibody dilution: Begin with 1:1000 dilution for HRP-conjugated antibodies and adjust based on signal strength .

• Loading controls: Use β-actin for total cellular protein extracts or COX IV for mitochondrial fractions .

• Detection system: SuperSignal West Pico or Femto Chemiluminescent Substrates provide sensitive detection of HRP-conjugated antibodies .

What considerations are important for immunohistochemical detection of TFF3?

For successful IHC applications with TFF3 antibodies:

• Antigen retrieval: Most protocols benefit from heat-induced epitope retrieval in citrate buffer (pH 6.0).

• Blocking: Use 1-5% BSA in TBS to minimize non-specific binding .

• Antibody concentration: Initial dilutions of 1:200-400 for IHC-P and 1:100-500 for IHC-F are recommended, with optimization based on tissue type .

• Visualization: For HRP-conjugated antibodies, DAB (3,3'-diaminobenzidine) substrate provides a stable, brown reaction product suitable for long-term storage.

• Counterstaining: Hematoxylin counterstaining provides nuclear context without interfering with the specific TFF3 signal.

How can TFF3 antibodies be used to investigate signaling pathways modulated by TFF3?

Research has revealed TFF3 involvement in multiple signaling pathways, which can be investigated using TFF3 antibodies in conjunction with other tools:

• NF-κB signaling: TFF3 activates NF-κB in intestinal epithelial cells. This can be studied by combining TFF3 antibody-based protein detection with electrophoretic mobility shift assays (EMSA) and supershift assays using antibodies against NF-κB subunits like p65 .

• ERK kinase pathway: TFF3 activates ERK kinase in intestinal epithelial cells, regulating Twist protein expression. This can be studied by using TFF3 antibodies in conjunction with inhibitors such as U0126 or PD-98059 .

• AKT signaling: Silencing TFF3 decreases phosphorylation of AKT-1 in prostate cancer cells. This relationship can be investigated by combining TFF3 antibody-based detection with phospho-specific AKT antibodies .

What approaches can be used to investigate the role of TFF3 in apoptotic regulation?

TFF3 has demonstrated anti-apoptotic functions in multiple contexts:

• Apoptotic protein expression: Western blot analysis using TFF3 antibodies alongside antibodies against apoptotic markers (cleaved caspase-3, cleaved caspase-9, cleaved PARP) can reveal relationships between TFF3 expression and apoptotic signaling .

• BAX/BCL2 ratio: Silencing TFF3 increases the BAX/BCL2 ratio at the mRNA level. This can be studied by combining TFF3 protein detection with qRT-PCR analysis of these apoptotic regulators .

• Subcellular fractionation: Combining TFF3 immunodetection with subcellular fractionation enables analysis of mitochondrial BAX translocation and release of proapoptotic proteins like cytochrome C and Smac/DIABLO in response to TFF3 modulation .

• Caspase-3 activity assays: Functional assessment of apoptotic signaling can be performed using colorimetric assays with the caspase-3 substrate (DEVD-ρNA) following TFF3 knockdown or overexpression .

What methodological approaches exist to investigate potential TFF3 receptor interactions?

Despite extensive research, TFF3 receptors remain incompletely characterized:

• Inositol 1 Phosphate (IP1) Assay: This HTRF-based competitive immunoassay can be used to investigate potential TFF3 interactions with proposed receptors like CXCR4, measuring fluorescence at 620 and 665 nm to calculate HTRF ratios that indicate receptor activation .

• Concentration considerations: When testing TFF3-receptor interactions, concentration ranges from 10^-12 to 10^-5 M should be considered to capture both high and low-affinity interactions .

• Positive controls: Include known ligands (e.g., CXCL12 for CXCR4) as positive controls to validate assay functionality .

• Antagonist vs. agonist activity: Differential experimental setups can distinguish between potential agonistic and antagonistic activities of TFF3 at its receptors .

How can I address inconsistent TFF3 detection in Western blot applications?

Inconsistent detection can arise from several sources:

• Protein degradation: TFF3 monomers are susceptible to gastrointestinal degradation, though more stable metabolites (TFF3 7-54) may retain bioactivity and trefoil structure . Use fresh samples with protease inhibitors and maintain cold conditions throughout extraction.

• Dimerization state: Native TFF3 can exist as monomers or homodimers through cysteine-mediated disulfide bonds, particularly via Cys57 . Reducing conditions may be necessary to obtain consistent monomeric detection.

• Glycosylation variations: Post-translational modifications may alter apparent molecular weight. Consider deglycosylation treatments to standardize migration patterns.

• Transfer efficiency: Small proteins like TFF3 may transfer inefficiently. Use PVDF membranes with appropriate pore size (0.2 μm) and optimize transfer conditions (20% methanol, 25V for 2 hours at 4°C) .

What control experiments should be included when studying TFF3's role in complex cellular processes?

Robust controls are essential for accurate interpretation:

• Positive and negative tissue controls: Include tissues with well-characterized TFF3 expression patterns, such as normal intestinal epithelia (positive) and skeletal muscle (negative).

• Recombinant protein standards: Include purified recombinant TFF3 as a molecular weight and antibody reactivity control .

• Multiple TFF3 antibodies: Validate key findings with multiple antibodies targeting different epitopes of TFF3 to confirm specificity .

• Gene silencing controls: Include both negative control siRNA and multiple TFF3-specific siRNAs to control for off-target effects in knockdown experiments .

What are the key considerations when comparing results from different experimental models expressing TFF3?

Inter-model comparison requires careful consideration:

• Expression level variability: TFF3 expression varies substantially between tissues and cell lines. Quantitative normalization to housekeeping genes is essential for meaningful comparisons .

• Dimerization differences: The monomer/homodimer ratio may vary between experimental systems, affecting function. Consider analyzing both forms when possible .

• Species-specific variations: Despite high conservation, minor sequence differences between human, mouse, and rat TFF3 may affect antibody recognition and functional outcomes. Use species-matched antibodies when possible .

• Subcellular localization: TFF3 localization varies between cytoplasmic, secreted, and extracellular matrix compartments. Use subcellular fractionation and appropriate markers to precisely define localization .

How can TFF3 antibodies contribute to understanding the role of TFF3 in inflammatory and immune responses?

Recent research highlights TFF3's immunomodulatory functions:

• Th17 cell responses: TFF3 may stimulate Th17 cell responses in type 2 diabetes mellitus, affecting IL-17A levels and RORγt/IL-17 signaling. This can be studied using flow cytometry with TFF3 and IL-17 antibodies .

• NF-κB modulation: TFF3 activates NF-κB differently from TNF, involving p65 homodimers rather than p50/p65 heterodimers. These differences can be explored using EMSA combined with supershift assays using specific antibodies .

• Twist protein regulation: TFF3 upregulates Twist protein expression in intestinal epithelial cells via ERK kinase. This relationship can be investigated using TFF3 antibodies together with ERK inhibitors like U0126 .

What methodological approaches can elucidate the role of TFF3 in cancer progression?

TFF3's role in cancer is an active research area:

• Proliferation assays: BrdU incorporation assays following TFF3 silencing or overexpression can quantify TFF3's impact on cancer cell proliferation .

• Migration analysis: Wound healing assays measuring cell migration distance using ImageJ software can assess TFF3's motogenic properties in different cancer contexts .

• Apoptotic resistance: Combined approaches using TFF3 antibodies with analyses of cleaved caspase-3, caspase-9, and PARP can reveal TFF3's anti-apoptotic functions in cancer cells .

• Mitochondrial integrity: Subcellular fractionation followed by Western blot analysis of BAX translocation, cytochrome C release, and Smac/DIABLO release can elucidate mechanisms of TFF3-mediated apoptotic resistance .

What are the considerations for developing and validating synthetic TFF3 peptides as research tools?

Synthetic TFF3 peptides offer unique research advantages:

• Chemical synthesis strategies: Native chemical ligation followed by oxidative folding enables production of both monomeric and homodimeric TFF3, allowing controlled analysis of each form .

• Structural validation: Correct folding of synthetic TFF3 should be confirmed by NMR and circular dichroism to ensure native-like structure and function .

• Metabolic stability assessment: Synthetic TFF3 and its homodimer are susceptible to gastrointestinal degradation, but gut-stable metabolites (e.g., TFF3 7-54) may retain bioactivity and should be characterized .

• Receptor validation: Synthetic TFF3 peptides can be used in receptor validation experiments, though studies with putative receptors like CXCR4 and LINGO2 have thus far shown negative results at concentrations up to 10 μM .