TGFB1I1 Antibody, HRP conjugated

What is TGFB1I1 and what cellular functions does it regulate?

TGFB1I1 (also known as HIC5 or ARA55) functions as a molecular adapter coordinating protein-protein interactions at focal adhesion complexes and in the nucleus. It links various intracellular signaling modules to plasma membrane receptors and regulates the Wnt and TGF-beta signaling pathways. In the nucleus, it serves as a nuclear receptor coactivator regulating glucocorticoid, androgen, mineralocorticoid, and progesterone receptor transcriptional activity. TGFB1I1 plays significant roles in cell growth, proliferation, migration, differentiation, and senescence processes . According to studies, HIC5/TGFB1I1 is 461 amino acids in length and contains four Leu:Asp-rich motifs and four LIM domains, with these domains performing distinct functions including nuclear matrix binding, coactivator activity, and focal adhesion binding .

What are the key specifications of commercially available TGFB1I1 antibodies?

Commercial TGFB1I1 antibodies vary in their specifications but typically include:

Most TGFB1I1 antibodies are generated using synthetic peptides or recombinant fusion proteins corresponding to specific amino acid sequences of human TGFB1I1, often targeting regions between amino acids 1-150 .

What does HRP conjugation contribute to TGFB1I1 antibody applications?

HRP (Horseradish Peroxidase) conjugation refers to the chemical attachment of the HRP enzyme to the antibody molecule. This modification eliminates the need for secondary antibody detection steps in assays such as Western blotting, ELISA, and immunohistochemistry. The HRP enzyme catalyzes a reaction with substrates to produce detectable signals (colorimetric, chemiluminescent), allowing direct visualization of the TGFB1I1 protein in experimental samples. HRP conjugation significantly reduces protocol time and potential background noise from secondary antibody cross-reactivity . Storage conditions for HRP-conjugated antibodies typically include buffered solutions with stabilizers such as glycerol and BSA, maintained at -20°C to preserve enzymatic activity .

How should Western blot protocols be optimized for HRP-conjugated TGFB1I1 antibodies?

For optimal Western blot results with HRP-conjugated TGFB1I1 antibodies, researchers should consider the following methodological approach:

• Sample preparation: Use complete lysis buffers containing protease inhibitors to prevent degradation

• Protein loading: Load 25-30 μg of total protein per lane for cell line lysates

• Dilution optimization: Start with a 1:500 to 1:2000 dilution range; validation data indicates 1:1000 as optimal for many cell types

• Blocking: Use 3% non-fat dry milk in TBST for 1 hour at room temperature

• Incubation time: Since HRP-conjugated antibodies eliminate the secondary antibody step, primary antibody incubation can be shorter (2-3 hours at room temperature)

• Detection system: Standard ECL detection provides sufficient sensitivity for TGFB1I1

• Expected molecular weight: Look for TGFB1I1 bands at approximately 50-55 kDa

• Positive controls: MCF-7, HepG2, and PC-3 human cell lines have been validated to express detectable TGFB1I1

What considerations are important for immunohistochemistry with TGFB1I1 antibodies?

When performing immunohistochemistry with TGFB1I1 antibodies, researchers should follow these methodological guidelines:

• Sample fixation: Both paraffin-embedded (IHC-P) and frozen sections (IHC-F) are compatible with TGFB1I1 antibodies

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer improves detection

• Antibody concentration: For HRP-conjugated antibodies, optimal dilutions range from 1:100 to 1:500

• Incubation parameters: 10 μg/mL for 3 hours at room temperature is effective for detecting TGFB1I1 in fixed cells

• Counterstaining: DAPI is recommended for nuclear visualization to assess subcellular localization

• Expected localization: TGFB1I1 shows both cytoplasmic and nuclear localization depending on cell stimulation state; unstimulated cells show primarily cytoplasmic localization while stimulated cells (e.g., with BMP-4) show stronger nuclear localization

• Controls: Include positive control tissues with known TGFB1I1 expression patterns

How can researchers validate the specificity of TGFB1I1 antibodies?

To rigorously validate TGFB1I1 antibody specificity, implement these methodological approaches:

• Positive controls: Use cell lines known to express TGFB1I1 such as MCF-7, HepG2, or PC-3

• Molecular weight verification: Confirm the detected band matches the expected molecular weight (50-55 kDa)

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding

• Cross-reactivity assessment: Test reactivity across multiple species if planning cross-species experiments; available antibodies show reactivity with human, mouse, rat, and other species

• Multiple antibodies approach: Compare results using antibodies targeting different epitopes of TGFB1I1

• Subcellular localization pattern: Verify that the observed localization matches known TGFB1I1 distribution patterns (cytoplasmic and nuclear, with translocation upon stimulation)

How can TGFB1I1 localization changes be effectively monitored in response to cellular stimuli?

TGFB1I1/HIC5 exhibits dynamic localization changes in response to stimuli. Research data indicates that in PC-3 cells, TGFB1I1 localization shifts from predominantly cytoplasmic (unstimulated) to nuclear (when stimulated with BMP-4) . To effectively monitor these changes:

• Experimental design: Set up parallel treatment groups with appropriate time points

• Stimulation protocol: Use defined concentrations of activators (e.g., 10 ng/mL BMP-4 or TGF-beta)

• Fixation method: Use paraformaldehyde fixation to preserve protein localization

• Antibody detection: For HRP-conjugated antibodies, use appropriate substrates; alternatively, fluorescent-conjugated secondary antibodies can be used with unconjugated primary TGFB1I1 antibodies

• Imaging: Confocal microscopy with z-stack acquisition provides optimal assessment of subcellular localization

• Quantification: Measure nuclear/cytoplasmic signal ratios across multiple cells and treatment conditions

• Controls: Include appropriate vehicle controls and time-matched untreated samples

What experimental design considerations are important when studying TGFB1I1's role in TGF-beta signaling?

When designing experiments to study TGFB1I1's role in TGF-beta signaling:

• Cell model selection: Choose cells responsive to TGF-beta that express TGFB1I1 (validated cell lines include MCF-7, HepG2, and PC-3)

• TGF-beta treatment parameters: TGF-beta is known to induce HIC5/TGFB1I1 expression

• Pathway activation verification: Monitor phosphorylation of SMAD proteins as positive controls

• Fractionation studies: Given TGFB1I1's dual localization in focal adhesions and nucleus, subcellular fractionation can help track its distribution

• Functional domains: Consider that TGFB1I1's LIM domains perform distinct functions (LIM4 binds to nuclear matrix, LIMs 3 and 4 act as coactivators, LIMs 2 and 3 bind to focal adhesions)

• Hydrogen peroxide effects: TGFB1I1/HIC5 is induced not only by TGF-beta but also by hydrogen peroxide, which may be relevant for oxidative stress studies

How can quantitative analysis of TGFB1I1 be performed in complex biological systems?

For quantitative assessment of TGFB1I1:

• Western blot quantification: Use HRP-conjugated TGFB1I1 antibodies with densitometry, loading 25 μg protein per lane

• Normalization strategy: Normalize to stable housekeeping proteins

• Subcellular fractionation: Separately quantify nuclear vs. cytoplasmic TGFB1I1 to assess distribution changes

• Fluorescence quantification: For immunofluorescence, NorthernLights™ 557-conjugated secondary antibodies have been validated for TGFB1I1 detection

• Dynamic range assessment: Create standard curves with recombinant TGFB1I1 protein to establish quantification limits

• Statistical analysis: Apply appropriate statistical tests when comparing TGFB1I1 levels across experimental conditions

How can non-specific binding be minimized when using HRP-conjugated TGFB1I1 antibodies?

To minimize non-specific binding:

• Antibody titration: Test multiple dilutions; recommended starting dilutions are 1:500-1:2000 for Western blot and 1:100-1:500 for IHC

• Blocking optimization: Extend blocking time with 3-5% BSA or non-fat milk in TBST

• For Western blots: Validated protocols use 3% non-fat dry milk in TBST for blocking

• For immunostaining: Adding 0.03% Proclin300 to buffers can help reduce background

• Storage buffer considerations: Commercial HRP-conjugated antibodies typically contain 1% BSA, 0.03% Proclin300, and 50% Glycerol in TBS (pH 7.4)

• Washing stringency: Increase washing duration and number of washes with TBST

• Antibody storage: Proper storage at -20°C and avoiding freeze-thaw cycles helps maintain specificity

What factors might lead to inconsistent results when using TGFB1I1 antibodies across different experiments?

When facing inconsistent results:

• Epitope accessibility: TGFB1I1 antibodies target different regions (N-terminal, middle region, C-terminal); epitope masking due to protein-protein interactions may occur

• Post-translational modifications: These can affect antibody binding

• Storage conditions: HRP activity can diminish over time with improper storage; aliquoting prevents repeated freeze-thaw cycles

• Cell type variations: TGFB1I1 expression and localization vary by cell type; MCF-7, HepG2, and PC-3 are validated positive controls

• Stimulation state: TGFB1I1 localization changes upon stimulation (e.g., with BMP-4 or TGF-beta)

• Sample preparation differences: Lysis buffers and fixation protocols can affect epitope availability

• Lot-to-lot antibody variability: Different manufacturing lots may show slight variations in specificity and sensitivity

How should researchers interpret contradictory results when different TGFB1I1 antibodies yield varying patterns?

When faced with contradictory results:

• Epitope mapping: Compare the epitopes recognized by each antibody; commercially available antibodies target different regions including amino acids 1-150, 138-200, 179-228, etc.

• Antibody format differences: Compare HRP-conjugated vs. unconjugated versions of the same antibody

• Validation methods: Review the validation data provided by manufacturers; Western blot validation in cell lines such as MCF-7 and HepG2 is documented for some antibodies

• Functional domain awareness: TGFB1I1's LIM domains have distinct functions; antibodies targeting different domains may detect different functional pools of the protein

• Literature concordance: Compare with published patterns and known biological functions of TGFB1I1

• Confirmatory approaches: Use RNA-level detection methods (RT-PCR, RNA-seq) to correlate with protein detection patterns

What emerging applications are being developed for TGFB1I1 antibodies in cancer research?

Emerging applications include:

• Biomarker development: TGFB1I1's role in cell migration and cancer progression makes it a potential prognostic marker

• Therapeutic target validation: As a regulator of TGF-beta signaling, TGFB1I1 may represent a therapeutic target

• Cellular plasticity studies: TGFB1I1's dynamic localization can serve as a marker for cell state transitions

• Nuclear receptor interaction studies: TGFB1I1 functions as a coactivator for multiple nuclear receptors

• Mechanotransduction research: TGFB1I1's role in focal adhesions makes it relevant for studying how cells respond to mechanical forces

How can multiplexed detection strategies be implemented to study TGFB1I1 in complex signaling networks?

For multiplexed detection:

• Sequential immunostaining: For HRP-conjugated antibodies, use sequential chromogenic detection with different substrates

• Panel design: Select markers that don't cross-react (focal adhesion proteins, nuclear receptors, signaling intermediates)

• Controls: Run single-stained controls to verify specificity

• Image acquisition: Capture images sequentially to minimize signal overlap

• Analysis considerations: Use software capable of co-localization analysis

• Positive control selection: PC-3 cells with and without BMP-4 stimulation provide a validated model for studying TGFB1I1 localization changes