TFF2 Antibody

What is TFF2 and why is it important in research?

Trefoil factor 2 (TFF2), also known as spasmolytic polypeptide (Sp), is a 20 kDa secreted protein belonging to the trefoil factor family. It is a protease-resistant glycoprotein primarily produced by mucous-secreting cells of the stomach and duodenum and is upregulated in response to inflammation. The 106-residue mature human TFF2 contains two trefoil domains (amino acids 36-72 and 80-121) and one N-linked glycosylation site . TFF2 interacts with mucin and other binding proteins to protect and repair mucosal epithelium, making it significant in research focused on gastrointestinal protection, inflammation, and cancer .

What are the common applications for TFF2 antibodies in research?

TFF2 antibodies are widely utilized in multiple laboratory techniques including:

• Western blot (WB) for protein detection in tissue lysates

• Immunohistochemistry (IHC) for tissue localization in both paraffin-embedded (IHC-p) and frozen sections

• Enzyme-linked immunosorbent assay (ELISA) for quantitative measurements

• Flow cytometry (FCM) for cell-specific expression analysis

Western blot analysis has successfully detected TFF2 in lysates of human stomach tissue, stomach cancer tissue, breast tissue, and kidney tissue with specific bands observed at approximately 19-21 kDa .

How should TFF2 antibodies be stored and handled to maintain optimal activity?

For optimal performance of TFF2 antibodies, researchers should follow these storage protocols:

• Store unopened antibody at -20°C to -70°C for up to 12 months from receipt date

• After reconstitution, store at 2-8°C under sterile conditions for up to 1 month

• For longer storage after reconstitution, aliquot and keep at -20°C to -70°C for up to 6 months

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles that can denature the antibody

What is the difference between polyclonal and monoclonal TFF2 antibodies?

Polyclonal TFF2 antibodies:

• Recognize multiple epitopes on the TFF2 protein

• Often produced in goats or rabbits against full-length or fragments of recombinant human TFF2

• Provide higher sensitivity but potentially lower specificity

• Example: Goat Anti-Human TFF2 Antigen Affinity-purified Polyclonal Antibody

Monoclonal TFF2 antibodies:

• Recognize a single epitope on the TFF2 protein

• Produced from a single B-cell clone, typically in mice or rats

• Offer higher specificity and more consistent lot-to-lot performance

• Example: Anti-TFF2 antibody [4G7C3] specifically optimized for Western blot and flow cytometry applications

How should TFF2 antibody be validated before use in experimental studies?

A robust validation protocol for TFF2 antibodies should include:

• Positive and negative control tissues:

  • Positive controls: Human stomach tissue (high expression)

  • Negative controls: Tissues known to lack TFF2 expression

• Western blot analysis to confirm:

  • Specific band at the expected molecular weight (19-21 kDa)

  • Absence of non-specific bands in negative control samples

• Concentration optimization:

  • Perform antibody titration (e.g., testing 0.5-5 µg/mL for Western blot)

  • The optimal concentration for Western blot appears to be around 2 μg/mL based on published protocols

• Cross-reactivity testing with related TFF family members (TFF1, TFF3) to ensure specificity

What are the optimal conditions for using TFF2 antibodies in Western blot analyses?

For optimal Western blot detection of TFF2:

• Sample preparation:

  • Use PVDF membrane (preferred over nitrocellulose for TFF2)

  • Conduct experiments under reducing conditions

• Antibody concentration:

  • 2 μg/mL of primary antibody (e.g., Goat Anti-Human TFF2 Antigen Affinity-purified Polyclonal Antibody)

• Secondary antibody:

  • HRP-conjugated Anti-Goat IgG Secondary Antibody

• Buffer system:

  • Immunoblot Buffer Group 1 has been reported to yield optimal results

• Expected molecular weight:

  • Look for specific bands at approximately 19-21 kDa

What are the considerations for using TFF2 antibodies in immunohistochemistry?

When using TFF2 antibodies for immunohistochemistry:

• Tissue preparation:

  • 4 μm thick formalin-fixed, paraffin-embedded sections

  • Proper deparaffinization and rehydration

• Antigen retrieval:

  • May be necessary due to formalin fixation masking epitopes

  • Method should be optimized for specific antibody

• Sectioning considerations:

  • Whole sections generally show better sensitivity than tissue microarrays (TMAs)

  • TMAs may result in false negatives due to heterogeneous TFF2 expression

  • In studies of gastric-type carcinoma, significantly fewer TFF2-positive cases were identified using TMAs compared to whole sections

• Interpretation guidelines:

  • Consider positive staining when cytoplasmic/membranous staining is observed in >5% of tumor cells

  • Note that TFF2 positive tumor cells may be present in limited areas, requiring extensive sampling

How can TFF2 antibodies be used to investigate its role in cancer research?

TFF2 antibodies have proven valuable in cancer research through several methodological approaches:

• Diagnostic marker development:

  • TFF2 has shown promise as a diagnostic marker for gastric-type carcinoma (GAS), the most common HPV-independent endocervical adenocarcinoma

  • When combined with HIK1083, double positivity is highly specific in separating GAS from non-GAS adenocarcinomas (p<0.01)

  • In whole section analysis, TFF2 was expressed in 80% (8/10) of GAS cases compared to only 12.5% (5/40) of non-GAS cases

• Tumor microenvironment studies:

  • TFF2 is expressed by macrophages and lymphocytes

  • Antibodies can help investigate TFF2's role in modulating immune system responses within the tumor microenvironment

• Therapeutic potential assessment:

  • TFF2 gene delivery via adenovirus (Ad-Tff2) has been shown to suppress colon tumor growth in colitis-associated cancer

  • Antibodies can assess expression levels following therapeutic interventions

What are the challenges in detecting TFF2 in different tissue samples?

Researchers face several methodological challenges when detecting TFF2 across tissue types:

• Heterogeneous expression patterns:

  • TFF2 positive tumor cells are often localized to limited areas of gastric-type adenocarcinomas

  • This requires extensive sampling to avoid false negatives

• Sampling method limitations:

  • TFF2 detection rate in gastric-type adenocarcinomas differs significantly between whole sections (80%) and tissue microarrays (29%)

  • Small biopsies may yield false negatives due to sampling error

• Cross-reactivity concerns:

  • TFF2 shares structural similarities with other trefoil factors

  • Careful antibody selection and validation is necessary to avoid cross-reactivity

• Tissue-specific expression levels:

  • High expression in stomach and duodenum

  • Lower expression in immune cells and colonic epithelium

  • Very low expression in colonic leukocytes compared to epithelial cells

How can researchers investigate the functional role of TFF2 using antibody-based approaches?

To investigate TFF2's functional roles, researchers can employ these antibody-based methodological strategies:

• Neutralizing antibody studies:

  • Use neutralizing TFF2 antibodies to block its function in cell culture or animal models

  • Assess the impact on inflammation, wound healing, or tumor growth

• Co-immunoprecipitation (Co-IP):

  • Identify TFF2-binding partners involved in mucosal protection

  • Characterize the TFF2 interactome in different physiological contexts

• ChIP-seq following TFF2 stimulation:

  • Identify transcriptional changes mediated by TFF2 signaling

  • Map the genomic landscape affected by TFF2 activity

• Immune cell phenotyping:

  • Use flow cytometry with TFF2 antibodies to characterize TFF2-expressing immune cell populations

  • Investigate how TFF2 deficiency alters immune cell populations and function

What strategies can address weak or absence of TFF2 immunostaining in tissue samples?

When encountering weak or absent TFF2 immunostaining, researchers should consider these methodological solutions:

• Sampling considerations:

  • Increase the number of tissue sections examined

  • Use whole sections rather than TMAs when possible

  • TFF2 positive tumor cells often appear in limited areas of specimens

• Antibody optimization:

  • Test different antibody concentrations (titration experiments)

  • Try alternative clones/vendors

  • Compare polyclonal versus monoclonal antibodies

• Antigen retrieval optimization:

  • Test different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize pH and buffer composition for epitope unmasking

• Detection system enhancement:

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

  • Use more sensitive detection reagents

• Technical controls:

  • Include known positive control tissues (stomach) in each experiment

  • Use an internal positive control within the same section when possible

How can researchers distinguish between specific and non-specific binding of TFF2 antibodies?

To ensure reliable distinction between specific and non-specific TFF2 antibody binding:

• Essential controls:

  • Negative tissue controls (tissues known to lack TFF2 expression)

  • Isotype controls to assess background from the antibody class

  • Blocking peptide competition assays to confirm binding specificity

• Signal validation approach:

  • Compare staining patterns across multiple independent TFF2 antibodies

  • Correlation with mRNA expression (ISH or RT-PCR)

  • Confirmation with genetic models (TFF2 knockout tissues as negative controls)

• Pattern recognition:

  • Specific TFF2 staining shows cytoplasmic/membranous localization

  • Expected molecular weight on Western blot is 19-21 kDa

  • Consider staining positive when present in >5% of target cells

What are the important considerations when using TFF2 antibodies for multiplexed immunofluorescence?

For successful multiplexed immunofluorescence studies including TFF2:

• Antibody panel design:

  • Select TFF2 antibodies raised in different host species from other targets

  • Ensure secondary antibodies lack cross-reactivity

  • Consider using directly conjugated primary antibodies to avoid secondary antibody conflicts

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include single-stained controls for spectral unmixing

  • Consider the relative expression levels when selecting fluorophores (brighter fluorophores for lower-expressed targets)

• Sequential staining protocol:

  • Test for epitope masking or destruction when using multiple antibodies

  • Consider tyramide signal amplification with sequential antibody stripping when using antibodies from the same species

• Validation of multiplexed results:

  • Compare multiplexed signals with single-antibody staining patterns

  • Confirm expected co-localization patterns based on known biology (e.g., TFF2 in stomach epithelial cells)

How should researchers interpret variations in TFF2 expression patterns across different disease states?

When analyzing TFF2 expression across disease states, researchers should consider:

• Baseline expression patterns:

  • High physiological expression in stomach and duodenum

  • Expression in immune cells (macrophages and lymphocytes)

  • Enrichment in colonic epithelial cells versus leukocytes

• Disease-specific alterations:

  • Upregulation during inflammation

  • Expression in 80% of gastric-type adenocarcinomas versus 12.5% of non-gastric-type adenocarcinomas

  • Potential alterations in cancer microenvironments

• Functional implications:

  • TFF2 plays roles in mucosal protection and repair

  • Has anti-inflammatory properties

  • May modulate immune system responses

  • Suppresses colon tumor growth in colitis-associated cancer

• Interpretation framework:

  • Consider heterogeneous expression when interpreting negative results

  • Use complementary markers (e.g., HIK1083) for confirming diagnoses

  • Take into account sampling limitations and methodological variables

What are the latest advances in using TFF2 antibodies for biomarker development?

Recent advances in TFF2 antibody applications for biomarker development include:

• Diagnostic applications:

  • TFF2 has emerged as a promising marker for gastric-type carcinoma (GAS)

  • When combined with HIK1083, double positivity provides high specificity for separating GAS from non-GAS adenocarcinomas

  • This biomarker approach addresses the aggressive behavior of GAS, the most common HPV-independent endocervical adenocarcinoma

• Modified TFF2 proteins for therapeutic development:

  • TFF2-CTP-Flag fusion proteins show improved pharmacokinetics while maintaining bioactivity

  • Such modifications address the poor natural pharmacokinetics of TFF2

  • These advancements enable exploration of TFF2 as a therapeutic agent for inflammatory conditions

• Predictive biomarker potential:

  • TFF2 expression patterns may predict response to therapies

  • TFF2 deficiency has been shown to exacerbate weight loss and alter immune cell populations

  • These findings suggest TFF2 as a potential biomarker for therapeutic response stratification

How can researchers integrate TFF2 antibody data with other molecular approaches for comprehensive pathway analysis?

For integrated pathway analysis incorporating TFF2 antibody data:

• Multi-omics data integration strategy:

  • Combine TFF2 protein expression data (immunohistochemistry/Western blot) with:

    • Transcriptomic data (RNA-seq, qPCR)

    • Genomic analyses (mutations, CNVs)

    • Epigenetic profiling (methylation, chromatin accessibility)

    • Interactome studies (co-IP, proximity labeling)

• Functional validation approaches:

  • Correlate TFF2 antibody staining with functional assays:

    • Cell migration/wound healing assays

    • Inflammatory response measurements

    • Mucosal integrity assessments

    • Gene knock-down/knock-out phenotypes

• Systems biology perspective:

  • Place TFF2 in the context of mucosal defense pathways

  • Map relationships between TFF2 and inflammatory mediators

  • Analyze TFF2's role in epithelial-immune cell crosstalk

  • Develop predictive models of TFF2 function in health and disease

• Translational research applications:

  • Connect TFF2 expression patterns with clinical outcomes

  • Evaluate TFF2 as a therapeutic target or biomarker

  • Assess the impact of TFF2-targeted interventions on disease progression