BAMBI Antibody

What is BAMBI and what are its key structural and functional properties?

BAMBI is a membrane-spanning glycoprotein that functions as a negative regulator of TGF-beta signaling during development. The human BAMBI protein:

• Has a molecular weight of approximately 29.1 kilodaltons

• Contains 260 amino acids

• Is also known as BMP and activin membrane-bound inhibitor homolog (Xenopus laevis), NMA, and non-metastatic gene A protein

• Acts as a pseudo-receptor that inhibits TGF-β signaling

BAMBI's transcriptional regulation is influenced by beta-catenin, BMP, smad3, and smad4. Functionally, BAMBI interferes with normal TGF-β receptor activation, thereby modulating downstream signaling pathways crucial for cellular differentiation, inflammation, and tissue repair processes .

What applications are BAMBI antibodies commonly used for in research?

BAMBI antibodies are utilized across multiple experimental techniques:

When performing these techniques, researchers should expect to detect BAMBI at approximately 29 kDa in appropriate samples .

What species reactivity do commercially available BAMBI antibodies exhibit?

Most commercially available BAMBI antibodies demonstrate cross-reactivity with multiple species:

When selecting an antibody for a specific species, verify the validation data provided by the manufacturer and consider preliminary validation experiments in your specific system .

How can I validate the specificity of a BAMBI antibody for my experimental system?

Comprehensive validation should include multiple approaches:

Positive Controls:

• Use cell lines with known BAMBI expression (based on literature)

• Include recombinant BAMBI protein as a standard

• Compare with tissues known to express BAMBI (e.g., kidney tubular epithelial cells)

Negative Controls:

• BAMBI knockout cells/tissues

• Samples treated with siRNA targeting BAMBI

• Secondary antibody-only controls

Orthogonal Validation:

• Confirm results with a second antibody targeting a different epitope

• Correlate protein expression with mRNA levels by qPCR

• Implement peptide blocking experiments using the immunizing peptide

The specificity validation should be performed for each application (WB, IHC, IF) separately, as antibody performance can vary between techniques .

What are the optimal conditions for using BAMBI antibodies in immunohistochemistry applications?

For successful IHC staining of BAMBI:

Tissue Preparation:

• Formalin-fixed, paraffin-embedded (FFPE) sections (3-5 μm)

• Fresh frozen sections also compatible with many antibodies

• Proper antigen retrieval is critical (typically heat-induced in citrate buffer pH 6.0)

Staining Protocol Optimization:

• Deparaffinize and rehydrate sections

• Perform antigen retrieval

• Block endogenous peroxidase (3% H₂O₂) and non-specific binding (5-10% normal serum)

• Incubate with primary antibody (optimal dilution determined empirically, often 1:100-1:500)

• Apply appropriate detection system (e.g., HRP-DAB)

• Counterstain, dehydrate, and mount

Published Example:
In a study examining BAMBI expression in human kidney, researchers successfully used a goat anti-human BAMBI/NMA antibody at 3 μg/mL overnight at 4°C, followed by HRP-DAB staining, which revealed specific cytoplasmic staining in tubular epithelial cells .

How does BAMBI expression vary across different tissue types and pathological conditions?

BAMBI demonstrates tissue-specific and disease-specific expression patterns:

Normal Tissues:

• Moderate expression in kidney tubular epithelial cells

• Variable expression in liver

• Expression in specific cell populations within the lung

Pathological Conditions:

• Hepatocellular Carcinoma (HCC): BAMBI has been identified as a prognostic biomarker associated with macrophage polarization, glycolysis, and lipid metabolism

• Psoriatic Arthritis: Altered BAMBI expression contributes to disease pathogenesis, making it a potential therapeutic target

• Chronic Obstructive Pulmonary Disease (COPD): BAMBI regulates macrophage function, inducing differentiation of Treg cells through the TGF-β pathway

• Chronic Wounds: Significantly increased expression (10.6-fold) compared to normal tissue, potentially inhibiting canonical TGF-β signaling

BAMBI expression should be quantified using standardized methods (e.g., H-score for IHC) and appropriate normalization for meaningful comparisons across different studies and tissue types.

How can BAMBI antibodies be used to investigate TGF-β signaling pathway disruption?

TGF-β signaling investigation using BAMBI antibodies requires multiparametric approaches:

Experimental Strategy:

• Dual Immunostaining: Co-localize BAMBI with TGF-β receptors to assess potential interference

• Phospho-SMAD Detection: Quantify downstream activation of canonical TGF-β signaling (p-SMAD2/3)

• Pathway Activity Assessment: Combine with reporter assays for TGF-β responsive elements

Methodological Example:
In chronic wound research, investigators employed:

• Western blotting to assess canonical TGF-β signaling components

• Immunostaining to visualize BAMBI and TGF-β pathway components in tissue sections

• TGF-β proteome profiler arrays to identify elevated BAMBI levels

• qPCR to quantify BAMBI expression in different wound types

• Reporter assays to confirm TGF-β activation status

This integrated approach revealed that despite TGF-β being activated, canonical signaling was decreased in chronic wounds, correlating with increased BAMBI expression (9.5-10.6 fold) .

What role does BAMBI play in immune cell function and how can this be studied?

BAMBI influences immune cell function through multiple mechanisms:

Key Findings:

• BAMBI promotes polarization of M1 macrophages

• BAMBI regulates the TGF-β/BAMBI pathway affecting Th17/Treg balance in COPD

• BAMBI expression correlates with immune cell infiltration in HCC

Experimental Approaches:

• Co-localization Studies: Immunofluorescence co-staining of BAMBI with immune cell markers (e.g., CD11c for dendritic cells)

• Functional Assays: Assessment of cytokine production and cell differentiation in the presence of BAMBI modulation

• In vivo Models: Evaluation of immune cell infiltration and function in BAMBI-modified animal models

Published Methodology:
Researchers investigating BAMBI's role in macrophage polarization used immunofluorescence staining with antibodies against CD11c (conjugated with Alexa Fluor 488, 1:100 dilution) and BAMBI (1:1000 dilution), followed by CY3-labeled secondary antibody (1:400) . Fluorescence intensity was quantified using ImageJ software to establish correlations between BAMBI expression and immune cell markers.

How is BAMBI being targeted therapeutically in inflammatory diseases?

BAMBI has emerged as a promising therapeutic target, particularly in inflammatory conditions:

Psoriatic Arthritis Therapy Development:
Inhibitec-Anticuerpos S.L. has developed a first-in-class humanized anti-BAMBI monoclonal antibody with a novel mechanism of action. This therapeutic approach:

• Targets BAMBI, a molecule not previously targeted in psoriasis treatments

• Impacts upstream TGF-β signaling intensity

• Creates a dual effect on the immune system by:

  • Reducing Th17 cells (main drivers of inflammation)

  • Increasing protective regulatory T cells that restrain inflammatory processes

• Shows potentially greater efficacy compared to current biologicals that only block specific pro-inflammatory mediators

The project received funding of $196,725 from the National Psoriasis Foundation to scale up production and conduct toxicology analyses prior to clinical trials .

What experimental models are best suited for studying BAMBI's role in disease progression?

Several experimental models have proven valuable for investigating BAMBI's functional roles:

In Vitro Models:

• Cell lines with manipulated BAMBI expression (overexpression/knockdown)

• Primary cell cultures from relevant tissues

• Co-culture systems to study cell-cell interactions

In Vivo Models:

• Mouse models of specific diseases:

  • Psoriasis and psoriatic arthritis models

  • Hepatocellular carcinoma models

  • Wound healing models

  • Pulmonary disease models

Experimental Design Example:
A study investigating BAMBI's role in tumor metastasis utilized:

• Tail vein injection of Huh7 cells with high BAMBI expression or control cells

• Monitoring for 11 weeks post-injection

• Analysis of lung and liver tissues using RT-qPCR and immunofluorescence

• Results showed that high-BAMBI-expression cells led to significantly increased tumor incidence compared to controls (two of six mice developed metastases versus none in the control group)

How do BAMBI expression levels correlate with clinical outcomes in cancer?

BAMBI has demonstrated value as a prognostic biomarker, particularly in hepatocellular carcinoma:

HCC Prognostic Associations:

• BAMBI expression correlates with pathological stage and nodal metastasis

• Associated with macrophage polarization, which influences tumor progression

• Connected to metabolic pathways including glycolysis and lipid metabolism

• Links to cell signaling pathways that drive cancer progression

Research Methods:
To establish these correlations, researchers employed:

• Pan-cancer analysis of BAMBI expression across multiple tumor types

• Correlation analyses between BAMBI levels and patient survival data

• Investigation of associations between BAMBI and pathological parameters

• Exploration of relationships between BAMBI and immune cell infiltration

• Prediction and analysis of genes interacting with BAMBI

What are the optimal storage and handling conditions for BAMBI antibodies?

Proper storage and handling are critical for maintaining antibody functionality:

Storage Recommendations:

• Store at -20°C to -70°C for long-term storage (up to 12 months from receipt)

• For short-term storage (up to 1 month), 2-8°C under sterile conditions after reconstitution

• Aliquot to avoid repeated freeze-thaw cycles

• Some formulations contain stabilizers (e.g., 0.02% sodium azide, 50% glycerol, pH 7.3)

Handling Best Practices:

• Centrifuge product if not completely clear after standing at room temperature

• Dilute only immediately before use

• For reconstituted antibodies, storage at -20°C to -70°C provides stability for up to 6 months

How can I optimize western blot conditions for detecting BAMBI protein?

Western blot optimization for BAMBI detection requires careful consideration of multiple parameters:

Sample Preparation:

• Use appropriate lysis buffers containing protease inhibitors

• Include phosphatase inhibitors if phosphorylation status is relevant

• Determine optimal protein loading (typically 20-50 μg total protein)

Electrophoresis and Transfer:

• Use 10-12% polyacrylamide gels for optimal resolution

• Consider using PVDF membranes for improved protein retention

• Verify transfer efficiency with reversible staining (e.g., Ponceau S)

Detection Parameters:

• Primary antibody dilution: Typically 1:500-1:2000 (optimize empirically)

• Incubation conditions: Often overnight at 4°C provides optimal results

• Secondary antibody selection: Match to host species of primary antibody

• Expected band size: Approximately 29 kDa

Published Examples:
Multiple studies have successfully detected BAMBI using western blot in various contexts, including TGF-β signaling studies in chronic wound tissues and analyses of BAMBI's role in adipogenesis .

What are common pitfalls when using BAMBI antibodies and how can they be addressed?

Researchers should be aware of several potential challenges:

Common Issues and Solutions:

Specific Recommendations:

• For IHC applications, optimize antigen retrieval methods as this significantly impacts BAMBI detection

• When studying tissues with variable BAMBI expression, include positive control tissues (e.g., kidney sections) to validate staining protocols

• Consider using multiple BAMBI antibodies targeting different epitopes to confirm specificity of observed patterns

How are BAMBI antibodies being used to investigate novel therapeutic approaches?

BAMBI antibodies are enabling several innovative therapeutic research directions:

Therapeutic Development Applications:

• Evaluation of humanized anti-BAMBI monoclonal antibodies for psoriatic arthritis

• Screening of compounds that modulate BAMBI expression or function

• Assessment of BAMBI's role in TGF-β-targeted therapies

• Investigation of BAMBI as a biomarker for patient stratification

Methodological Approaches:

• Preclinical model testing using anti-BAMBI antibodies

• Pharmacological characterization of antibody-based therapeutics

• Toxicology studies to establish safety profiles

• Biomarker development correlating BAMBI levels with treatment response

The ongoing research on humanized anti-BAMBI monoclonal antibodies represents a first-in-class approach that targets upstream signaling mechanisms rather than downstream inflammatory mediators, potentially offering advantages over current biological treatments for inflammatory conditions .

What new insights have been gained about BAMBI's role in cellular metabolism?

Recent research has revealed unexpected connections between BAMBI and metabolic processes:

Key Metabolic Associations:

• BAMBI has been linked to glycolysis regulation in hepatocellular carcinoma

• Connections between BAMBI expression and lipid metabolism have been identified

• BAMBI may influence metabolic reprogramming during disease progression

Research Approaches:

• Metabolomic profiling in systems with manipulated BAMBI expression

• Correlation analyses between BAMBI levels and metabolic gene signatures

• Functional studies examining the impact of BAMBI modulation on metabolic pathways

These emerging connections suggest BAMBI may have broader regulatory roles beyond TGF-β signaling, potentially influencing cellular energy utilization and metabolic adaptation during disease states .

What are promising future applications of BAMBI antibodies in translational medicine?

Several promising translational applications are emerging:

Potential Clinical Applications:

• Development of diagnostic assays for BAMBI expression in tissue biopsies

• Use as companion diagnostics for TGF-β pathway-targeted therapies

• Monitoring treatment response in inflammatory conditions

• Prognostic stratification in cancer patients

Research Needs:

• Standardization of BAMBI detection methods across laboratories

• Establishment of clinically relevant thresholds for BAMBI expression

• Validation in larger patient cohorts across multiple disease settings

• Integration with other biomarkers for comprehensive pathway analysis

These applications could help bridge the gap between basic research findings and clinical implementation, potentially improving patient stratification and treatment selection .

How might single-cell analysis techniques enhance our understanding of BAMBI function?

Single-cell technologies offer new opportunities for investigating BAMBI:

Analytical Approaches:

• Single-cell RNA sequencing to identify cell populations with dynamic BAMBI expression

• Mass cytometry for simultaneous detection of BAMBI and multiple signaling components

• Spatial transcriptomics to map BAMBI expression within tissue architecture

• Live-cell imaging with fluorescently tagged antibodies to track BAMBI trafficking

Research Questions Addressable:

• How does BAMBI expression vary among individual cells within a tissue?

• Which cell types show the most dynamic regulation of BAMBI during disease progression?

• How does cellular heterogeneity in BAMBI expression influence tissue-level responses?

• What spatial relationships exist between BAMBI-expressing cells and their microenvironment?