TGFB1I1 Antibody, FITC conjugated

What is TGFB1I1 and why is it important in cellular research?

TGFB1I1 (Transforming growth factor beta-1-induced transcript 1 protein) functions as an androgen receptor coactivator (55 kDa protein) and is also known as hydrogen peroxide-inducible clone 5 protein (Hic-5). This protein plays critical roles in TGF-β1 signaling pathways and androgen receptor function, making it relevant for research in cancer biology, fibrosis, and cellular adaptation to stress. Methodologically, studying TGFB1I1 requires specific antibodies like the FITC-conjugated variant to visualize its expression and localization within cellular compartments . The protein's dual role in both androgen signaling and TGF-β pathways makes it a significant intersection point for understanding complex cellular regulatory networks.

What experimental applications are suitable for TGFB1I1 Antibody, FITC conjugated?

• Flow cytometry: The FITC conjugation makes this antibody suitable for direct detection in flow cytometry without secondary antibodies, similar to the approach used with other FITC-conjugated antibodies in immunophenotyping studies .

• Fluorescence microscopy: The direct fluorescent labeling enables visualization of TGFB1I1 localization in fixed cells.

• Immunohistochemistry: With appropriate optimization, researchers can use this for tissue section analysis with fluorescent detection.

When designing experiments, ensure proper controls are included to account for potential background fluorescence and validate specificity through appropriate blocking experiments.

What are the optimal storage conditions for maintaining TGFB1I1 Antibody, FITC conjugated activity?

For maximum retention of activity, store TGFB1I1 Antibody, FITC conjugated at -20°C or -80°C upon receipt . Methodologically, it's crucial to avoid repeated freeze-thaw cycles, which can degrade both the antibody protein and the FITC fluorophore. The antibody is supplied in a protective buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When working with the antibody:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Protect from light exposure during handling and storage to prevent photobleaching of the FITC fluorophore

• When removing from storage, allow the antibody to equilibrate to room temperature before opening to prevent moisture condensation

The liquid form with glycerol facilitates storage stability, but proper handling remains essential for maintaining consistent experimental results.

How should I determine the optimal dilution for TGFB1I1 Antibody, FITC conjugated in flow cytometry applications?

When optimizing TGFB1I1 Antibody, FITC conjugated for flow cytometry, a systematic titration approach is necessary. Drawing from similar FITC-conjugated antibody methodologies:

• Begin with a titration series using serial dilutions (e.g., 0.5 μL, 1 μL, and 5 μL of 1 mg/mL antibody solution per 10^6 cells)

• Evaluate signal-to-noise ratio at each concentration

• Select the concentration providing adequate separation between positive and negative populations without excessive background

As demonstrated in similar antibody applications, 0.5 μL of FITC-conjugated antibody at 1 mg/mL concentration can provide optimal signals, while higher volumes (1 μL and 5 μL) may result in out-of-scale signals . The optimal concentration should be determined empirically for each application and cellular system.

What controls should be included when using TGFB1I1 Antibody, FITC conjugated?

Rigorous control implementation is essential for accurate interpretation of results with TGFB1I1 Antibody, FITC conjugated:

• Isotype control: Use FITC-conjugated rabbit IgG at the same concentration to assess non-specific binding

• Unstained controls: Essential for setting baseline fluorescence and adjusting for autofluorescence

• Blocking controls: Pre-incubation with recombinant TGFB1I1 protein should diminish specific staining

• Positive controls: Cell lines with known TGFB1I1 expression levels

• Negative controls: Cell lines with minimal or no TGFB1I1 expression

For flow cytometry applications specifically, a fluorescence-minus-one (FMO) control helps distinguish positive populations from spectral overlap when using multiple fluorophores. This methodological approach ensures reliable differentiation between specific and non-specific signals .

How can I verify the specificity of TGFB1I1 Antibody, FITC conjugated?

Verification of antibody specificity requires multiple complementary approaches:

• Western blot validation: Prior to fluorescent applications, confirm specificity using the non-conjugated version of the antibody to detect a single band at the expected molecular weight (~55 kDa for TGFB1I1)

• Competitive binding assay: Pre-incubate the antibody with purified recombinant TGFB1I1 protein (138-200AA region is particularly relevant as it contains the immunogen sequence)

• Genetic validation: Compare staining between TGFB1I1 knockdown/knockout and wild-type cells

• Cross-reactivity assessment: Test against related proteins, particularly other TGF-β pathway components

Methodologically, maintaining consistent experimental conditions across these validation steps is crucial for meaningful comparison and confident confirmation of specificity.

How can TGFB1I1 Antibody, FITC conjugated be used to study TGF-β1 signaling pathways?

TGFB1I1 Antibody, FITC conjugated offers sophisticated approaches to investigate TGF-β1 signaling dynamics:

• Time-course analysis: Monitor TGFB1I1 expression and subcellular localization changes following TGF-β1 stimulation using flow cytometry or confocal microscopy

• Co-localization studies: Combine with antibodies against other TGF-β pathway components (using different fluorophores) to examine protein-protein interactions

• Cell-type specific expression: Flow cytometric analysis of TGFB1I1 expression in diverse cell populations to identify differential responses to TGF-β1

This methodological approach can be particularly valuable in understanding how microbiota and environmental factors influence TGF-β1 signaling, similar to studies that have demonstrated microbiota-dependent regulation of TGF-β1 expression in regulatory T cells . By measuring TGFB1I1 levels in different cellular contexts, researchers can gain insights into the downstream effects of TGF-β1 signaling perturbations.

What role does TGFB1I1 play in regulatory T cell function and how can this be studied using FITC-conjugated antibodies?

TGFB1I1, as a TGF-β1-induced protein, may be implicated in regulatory T cell (Treg) functions, particularly given the critical role of TGF-β1 in Treg development and immunosuppressive activity. Recent research has demonstrated that Treg-derived TGF-β1 controls multiple immune checkpoints in a gene dose and microbiota-dependent manner .

Methodological approaches for studying TGFB1I1 in Treg contexts include:

• Multi-parameter flow cytometry: Combine TGFB1I1 Antibody, FITC conjugated with markers for Treg cells (CD4, CD25, FOXP3) using different fluorophores

• Functional correlation analysis: Sort Treg cells based on TGFB1I1 expression levels and assess their suppressive capacity in vitro

• Context-dependent expression: Compare TGFB1I1 expression in Tregs from different microbiota contexts (e.g., specific pathogen-free vs. germ-free conditions)

This sophisticated approach permits investigation of whether TGFB1I1 serves as a molecular bridge between TGF-β1 signaling and the regulatory functions of Tregs, particularly in contexts where microbiota influence immune tolerance.

How can TGFB1I1 Antibody, FITC conjugated be used in conjunction with other techniques to study protein-protein interactions?

Combining TGFB1I1 Antibody, FITC conjugated with complementary techniques enables comprehensive analysis of protein-protein interactions:

• Fluorescence Resonance Energy Transfer (FRET): Pair FITC-conjugated TGFB1I1 antibody with antibodies against potential binding partners conjugated to compatible FRET acceptor fluorophores

• Proximity Ligation Assay (PLA): Use the antibody in combination with PLA probes to detect protein interactions with high sensitivity

• Co-immunoprecipitation followed by flow cytometry: Perform co-IP using anti-TGFB1I1 antibody, then stain with FITC-conjugated TGFB1I1 antibody to confirm pull-down efficiency

• Imaging flow cytometry: Combine the spatial resolution of microscopy with the high-throughput capabilities of flow cytometry to visualize protein co-localization

This methodological integration allows researchers to not only identify interaction partners but also characterize the subcellular compartments where these interactions occur and how they change under different stimulation conditions or disease states.

What factors might contribute to weak or absent signals when using TGFB1I1 Antibody, FITC conjugated?

Several methodological issues can lead to suboptimal signal detection:

• Fluorophore degradation: FITC is susceptible to photobleaching. Minimize light exposure during handling and consider using anti-fade mounting media for microscopy applications.

• Protein denaturation: Improper storage or handling may compromise epitope recognition. Verify antibody functionality using positive control samples.

• Insufficient permeabilization: For intracellular targets, optimize permeabilization conditions to ensure antibody access while maintaining cellular integrity.

• Low target expression: TGFB1I1 expression may vary with cell type and activation state. Consider using positive control cell types with known expression.

• Buffer incompatibility: The antibody is formulated in a specific buffer (50% Glycerol, 0.01M PBS, pH 7.4) ; significant deviations from compatible buffer systems may affect binding.

Systematic troubleshooting requires changing one variable at a time while maintaining appropriate controls to identify the specific factor limiting detection.

How can I minimize background fluorescence when using TGFB1I1 Antibody, FITC conjugated in microscopy or flow cytometry?

Controlling background fluorescence requires attention to several methodological aspects:

• Block adequately: Use 1-3% BSA or 5-10% serum from the same species as the secondary antibody (if used) for at least 30 minutes

• Optimize antibody concentration: Excessive antibody concentrations increase non-specific binding; refer to titration results for optimal dilution

• Include proper washes: Multiple washes with PBS containing 0.05-0.1% Tween-20 help remove unbound antibody

• Address autofluorescence:

  • For flow cytometry: Use unstained controls and adjust compensation accordingly

  • For microscopy: Consider treatments to reduce autofluorescence (e.g., Sudan Black B for tissues)

• Filter samples adequately: Remove cell clumps and debris before analysis, as these often contribute to non-specific fluorescence

Implementing these methodological refinements systematically can significantly improve signal-to-noise ratio in fluorescence-based applications.

What factors should be considered when using TGFB1I1 Antibody, FITC conjugated in multiplex fluorescence applications?

Successful multiplex fluorescence experiments with TGFB1I1 Antibody, FITC conjugated require attention to several technical considerations:

• Spectral overlap: FITC (excitation ~495 nm, emission ~519 nm) may overlap with other fluorophores. Design panels carefully considering available instrumentation:

• Fixation effects: Some fixation methods can affect FITC fluorescence intensity. Paraformaldehyde (1-4%) is generally compatible with FITC conjugates.

• Antibody panel design: When combining multiple antibodies, consider:

  • Abundance of targets (use brighter fluorophores for less abundant targets)

  • Antigen sensitivity to fixation and permeabilization

  • Potential antibody cross-reactivity

• Sequential staining: For complex panels, consider sequential rather than simultaneous staining to minimize interference.

This methodological approach ensures reliable multiplex detection while minimizing artifacts from fluorophore interactions or processing effects.

How should I design experiments to study TGFB1I1 interactions with the androgen receptor using FITC-conjugated antibodies?

TGFB1I1 functions as an androgen receptor coactivator (ARA55) , necessitating specialized experimental approaches:

• Co-localization studies: Use TGFB1I1 Antibody, FITC conjugated alongside androgen receptor antibodies conjugated to spectrally distinct fluorophores to visualize potential co-localization

• Hormone stimulation experiments:

  • Treat cells with dihydrotestosterone (DHT) or other androgens

  • Monitor temporal changes in TGFB1I1 localization using the FITC-conjugated antibody

  • Compare cytoplasmic versus nuclear distribution before and after stimulation

• Functional correlation analysis:

  • Sort cells based on TGFB1I1 expression levels using the FITC-conjugated antibody

  • Assess androgen receptor transcriptional activity in sorted populations

• Proximity-based assays: Implement FRET or PLA using the FITC-conjugated antibody to directly assess physical interactions between TGFB1I1 and androgen receptor

This methodological integration provides multi-dimensional insights into how TGFB1I1 participates in androgen receptor signaling across different cellular contexts and stimulation conditions.

What considerations are important when designing experiments to study TGFB1I1 expression in different tissue and cell types?

Comprehensive analysis of TGFB1I1 expression patterns requires attention to several methodological aspects:

• Sample preparation optimization:

  • Cell suspensions: Different tissues require specific dissociation protocols to maintain epitope integrity

  • Fixed tissues: Optimize fixation, antigen retrieval, and permeabilization for the specific tissue type

• Expression baseline establishment:

  • Create a reference panel of tissues/cell types with known TGFB1I1 expression levels

  • Include both positive (high expression) and negative (minimal expression) controls

• Context-dependent expression analysis:

  • Consider cell activation states (resting vs. stimulated)

  • Assess expression under different microenvironmental conditions, which may affect TGF-β pathway activity

• Validation through complementary approaches:

  • Flow cytometry for quantitative expression analysis

  • Immunofluorescence microscopy for spatial distribution

  • mRNA analysis (e.g., qPCR, RNA-seq) to correlate protein with transcript levels

This multi-faceted approach ensures reliable characterization of TGFB1I1 expression patterns across diverse biological contexts.

How can I use TGFB1I1 Antibody, FITC conjugated to study the relationship between TGF-β1 signaling and microbiota?

Recent research has revealed important connections between TGF-β1 signaling and microbiota composition . To investigate these relationships using TGFB1I1 Antibody, FITC conjugated:

• Comparative analysis across microbiota states:

  • Compare TGFB1I1 expression in samples from germ-free versus specific pathogen-free animals

  • Assess changes following colonization with defined bacterial consortia (e.g., Clostridiales species)

• Cell-type specific responses:

  • Use multi-parameter flow cytometry to identify which cell populations modulate TGFB1I1 expression in response to microbiota

  • Focus particularly on immune cell populations in gut-associated lymphoid tissues

• Temporal dynamics:

  • Monitor TGFB1I1 expression changes during microbial colonization or following antibiotic treatment

  • Correlate with TGF-β1 expression and activity

• Functional validation:

  • Sort cells based on TGFB1I1 expression levels using the FITC-conjugated antibody

  • Assess their functional properties in immunological assays

This methodological approach aligns with recent findings demonstrating microbiota-dependent regulation of TGF-β1 expression in specific immune cell populations, particularly regulatory T cells .