TGFB1I1 Antibody, Biotin conjugated

Basic Research Questions

• What is TGFB1I1 and what cellular functions does it regulate?

  TGFB1I1 (Transforming growth factor beta-1-induced transcript 1) is a protein encoded by the TGFB1I1 gene in humans. It was originally isolated as a senescence-inducing gene from mouse osteoblastic cells through treatment with transforming growth factor beta-1 and hydrogen peroxide . TGFB1I1 plays significant roles in multiple cellular processes including:

  • Cell growth and proliferation

  • Cell migration and adhesion

  • Cellular differentiation

  • Senescence

  TGFB1I1 is primarily localized at focal adhesion complexes of cells, although it may also be found active in the cytosol, nucleus, and cell membrane . The protein has been characterized as a focal adhesion protein, an androgen and glucocorticoid receptor co-activator, and a negative regulator of muscle differentiation .

• What are the key applications for biotin-conjugated TGFB1I1 antibodies?

  Biotin-conjugated TGFB1I1 antibodies are versatile tools validated for multiple research applications:

  Application	Usage Notes
  Western Blotting (WB)	Validated at concentrations of 0.25-0.5 μg/ml
  ELISA	Effective at concentrations of 0.1-0.5 μg/ml
  Immunohistochemistry (IHC)	Used for both paraffin-embedded (IHC-P) and frozen sections (IHC-F)
  Immunocytochemistry (ICC)	Validated for cellular localization studies

  The biotinylation allows for amplified signal detection when used with streptavidin-conjugated detection systems, enhancing sensitivity in various assays .

• How do TGFB1I1 and TGFB1 differ, and why is this distinction important for antibody selection?

  Though related, these proteins have distinct functions and structures:

  • TGFB1 (Transforming Growth Factor Beta 1): A multifunctional cytokine that regulates proliferation, differentiation, adhesion, and migration in many cell types . TGFB1 exists in both latent and active forms.

  • TGFB1I1: A transcript induced by TGFB1 signaling . It functions primarily as a focal adhesion protein and transcriptional co-regulator.

  When selecting antibodies, researchers must ensure they're targeting the correct protein. Antibodies against TGFB1 detect the growth factor itself , while TGFB1I1 antibodies detect the induced protein . Using the wrong antibody would yield completely different biological insights and potentially misleading results.

• What species reactivity has been validated for commercially available TGFB1I1 antibodies?

  Available TGFB1I1 antibodies demonstrate wide cross-species reactivity:

  Species	Validated Reactivity	Reference
  Human	Yes - 100% sequence homology
  Mouse	Yes - 100% sequence homology
  Rat	Yes - 100% sequence homology
  Cow	Yes - 100% predicted reactivity
  Dog	Yes - 93% predicted reactivity
  Rabbit	Yes - 100% predicted reactivity
  Horse	Yes - 100% predicted reactivity
  Goat	Yes - 86% predicted reactivity
  Guinea Pig	Yes - 100% predicted reactivity
  Monkey	Demonstrated in COS-7 cells

  This broad cross-reactivity makes these antibodies versatile tools for comparative studies across species .

Advanced Research Applications

• What are the optimal Western blot conditions when using biotin-conjugated TGFB1I1 antibodies?

  For optimal Western blot detection of TGFB1I1, the following conditions have been experimentally validated:

  • Sample preparation: Use 30 μg of sample under reducing conditions

  • Gel conditions: 5-20% SDS-PAGE gel at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

  • Transfer conditions: Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Blocking: 5% non-fat milk in TBS for 1.5 hours at room temperature

  • Primary antibody: Biotin-conjugated TGFB1I1 antibody at 0.5 μg/mL incubated overnight at 4°C

  • Washing: TBS with 0.1% Tween, 3 washes of 5 minutes each

  • Detection system: Streptavidin-HRP followed by enhanced chemiluminescence

  • Expected band size: Approximately 50-75 kDa (specific band detected at ~50 kDa in R&D Systems validation and ~75 kDa in Boster Bio validation)

• How should researchers design immunohistochemistry experiments with TGFB1I1 biotin-conjugated antibodies?

  For successful immunohistochemistry with biotin-conjugated TGFB1I1 antibodies:

  1. Tissue preparation: Both paraffin-embedded (IHC-P) and frozen sections (IHC-F) are suitable

  2. Antigen retrieval: Critical for paraffin sections; heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

  3. Antibody dilutions:

     • For IHC-P: 1:200-400 dilution

     • For IHC-F: 1:100-500 dilution

  4. Controls:

     • Positive control: Use PC-3 human prostate cancer cell line, which has shown consistent TGFB1I1 expression

     • Negative control: Omit primary antibody or use isotype control

  5. Subcellular localization: Expect different patterns based on cellular activation state:

     • Unstimulated cells: Primarily cytoplasmic localization

     • Stimulated cells (e.g., with BMP-4): Nuclear translocation observed

  6. Signal detection: Use streptavidin-conjugated fluorophores (like NorthernLights 557) for fluorescent detection or streptavidin-HRP with chromogenic substrates for brightfield microscopy

• What approaches are recommended for validating the specificity of TGFB1I1 antibodies?

  Comprehensive validation of TGFB1I1 antibodies should include:

  1. Western blot validation: Confirm single band at expected molecular weight (~50-75 kDa) in positive control samples (MCF-7, HepG2, PC-3, or COS-7 cell lines)

  2. Peptide competition assay: Pre-incubate antibody with immunizing peptide before application to verify signal elimination

  3. Cell line panel screening: Test antibody across multiple cell lines with known differential expression of TGFB1I1

  4. Knockdown validation: Compare antibody reactivity in wild-type versus TGFB1I1 siRNA-treated samples

  5. Cross-species reactivity: Verify performance across species if doing comparative studies, noting potential differences in signal intensity even with high sequence homology

  6. Cell stimulation: Compare subcellular localization in unstimulated versus stimulated conditions (e.g., BMP-4 treatment should induce nuclear translocation)

• What methods are most effective for quantifying TGFB1I1 protein using biotin-conjugated antibodies in ELISA?

  For optimal ELISA quantification of TGFB1I1:

  1. Sandwich ELISA approach:

     • Use capture antibody specific for TGFB1I1 pre-coated onto microplate

     • Apply standards and samples

     • Add biotin-conjugated detection antibody at 0.1-0.4 μg/ml

     • Apply streptavidin-HRP conjugate

     • Develop with appropriate substrate and measure signal

  2. Sample types validated for TGFB1I1 detection:

     • Cell culture supernatants

     • Plasma

     • Serum

     • Other biological fluids

  3. Assay optimization considerations:

     • Working time: Typically 3-5 hours for standard sandwich ELISA

     • Antibody concentration: 0.1-0.5 μg/ml for detection antibody

     • Blocking agents: Use protein-free blockers to avoid background when using biotin-conjugated antibodies

     • Signal amplification: Avidin-biotin complexes (ABC) can further enhance sensitivity

• How can researchers distinguish between nuclear and cytoplasmic TGFB1I1 in experimental systems?

  TGFB1I1 undergoes nucleocytoplasmic shuttling in response to stimuli. To accurately distinguish between compartments:

  1. Immunofluorescence approach:

     • Fix cells using paraformaldehyde (4%) to preserve subcellular structures

     • Use biotin-conjugated TGFB1I1 antibody (10 μg/mL) optimized for ICC

     • Counterstain nuclei with DAPI

     • Visualize using streptavidin-conjugated fluorophores (e.g., NorthernLights 557)

     • Analyze using confocal microscopy for precise subcellular localization

  2. Subcellular fractionation approach:

     • Separate nuclear and cytoplasmic fractions using established protocols

     • Confirm fraction purity using compartment-specific markers (e.g., histone H3 for nuclear, GAPDH for cytoplasmic)

     • Perform Western blot using biotin-conjugated TGFB1I1 antibody

     • Quantify relative distribution between compartments

  3. Stimulation conditions affecting localization:

     • Unstimulated cells: Predominantly cytoplasmic localization

     • BMP-4 stimulation (10 ng/mL): Induces nuclear translocation

     • TGF-β1 treatment: Can alter subcellular distribution

     • Oxidative stress (H₂O₂): May affect hydrogen peroxide-inducible localization

• What considerations are important when using TGFB1I1 antibodies to study protein-protein interactions?

  When investigating TGFB1I1 protein interactions:

  1. Co-immunoprecipitation approach:

     • Choose antibody binding region carefully to avoid disrupting interaction domains

     • Middle region antibodies (AA 256-461) may be suitable for many interactions

     • Consider biotinylated antibodies for gentle elution with biotin competition

  2. Known interaction partners to consider:

     • TGFB1I1 interacts with various proteins including androgen receptor

     • Consider positive controls based on established interactions

  3. Validation controls:

     • Input control: 5-10% of lysate used for IP

     • Negative control: IgG from same species as primary antibody

     • Reciprocal IP: Confirm interaction by IP with antibody against suspected partner

  4. Detection strategy:

     • For biotin-conjugated antibodies: Use streptavidin-conjugated reporter systems

     • Consider streptavidin magnetic beads for IP when using biotinylated antibodies

     • Avoid biotin-containing buffers or sera that may interfere with biotin-streptavidin interaction

• What are the key experimental considerations when using TGFB1I1 autoantibodies as potential disease biomarkers?

  Recent research has identified anti-TGFB1I1 autoantibodies as potential biomarkers:

  1. Clinical applications:

     • Anti-TGFB1I1 autoantibodies show correlation with certain disease states

     • Significant association with sex-specific patterns has been observed

  2. Detection methodology:

     • Use whole-proteome arrays (WPAs) with GST-tagged TGFB1I1 proteins

     • Dilute human serum 3:1000 in reaction buffer containing synthetic block, PBS, and 0.1% Tween 20

     • Apply goat anti-Human IgG (H+L) Alexa Fluor 647 conjugate at 1:1000 dilution

     • Quantify using fluorescence imaging

  3. Data analysis considerations:

     • Account for potential cross-reactivity with other autoantibodies

     • Perform correlation analysis (using Spearman's r > 0.5 as threshold)

     • Consider sequence alignment and identity analysis for proteins showing correlation

     • Analyze sex-specific distributions separately

  4. Control selection:

     • Include healthy, age-matched controls

     • Consider both disease-specific and general inflammatory controls

• How can researchers optimize experiments studying TGFB1I1 in the context of TGF-β1 signaling?

  To effectively study TGFB1I1 in TGF-β1 signaling pathways:

  1. Experimental design considerations:

     • Include both TGFB1 and TGFB1I1 detection in parallel

     • Use cell models with validated TGF-β receptor expression

     • Consider time-course experiments to capture induction kinetics

  2. Stimulation protocols:

     • TGF-β1 treatment: Typically 1-10 ng/mL for 24-72 hours

     • BMP-4 stimulation: 10 ng/mL has been validated for TGFB1I1 translocation studies

     • Compare effects of active vs. latent TGF-β1 forms

  3. Important controls:

     • TGF-β receptor inhibitors to confirm signaling specificity

     • TGFB1I1 knockdown/knockout models to confirm functional relevance

     • Antibodies targeting specific TGF-β1 forms (e.g., LTBP-complexed TGF-β1)

  4. Detection strategy:

     • Monitor both protein levels (Western blot/ELISA) and subcellular localization (ICC/IF)

     • Consider using biotin-conjugated antibodies against both TGFB1 and TGFB1I1

     • Implement multicolor imaging to simultaneously track both proteins