BMP8B Antibody

What is BMP8B and what distinguishes it from other BMP family members?

BMP8B (Bone morphogenetic protein 8b, also known as OP-3) is a member of the Gbb-60A class of the BMP family within the TGF-beta superfamily. Unlike some BMPs that primarily function in bone formation, BMP8B plays specialized roles in reproductive development and metabolic regulation. The protein functions in cartilage development and cell differentiation, with critical regulatory roles in embryonic development . In mouse models, BMP8B has been shown to promote BMP-4 signaling through repressive action on the anterior visceral endoderm, influencing germ cell lineage commitment in early development .

What is the molecular structure and characteristics of BMP8B?

BMP8B is synthesized as a preproprecursor protein of 399 amino acids in mouse (402 in human), consisting of a 19 amino acid signal sequence, 241 amino acid prosegment, and a 139 amino acid mature region . The mature protein forms a functional homodimer through an interchain disulfide bond at Cys363 . The expected molecular weight of the mature BMP8B is approximately 16 kDa, while the precursor form is detected at approximately 45 kDa in Western blot analysis . Mouse BMP8B shares 74% amino acid identity with mouse BMP8A, 69% identity with human BMP8B, and 93% identity with rat BMP8B over amino acids 261-399 .

How do BMP8A and BMP8B differ in humans and mice?

In both humans and mice, BMP8A and BMP8B exist as two closely linked but distinct genes. Human BMP8A and BMP8B protein products share 98% amino acid sequence identity, making their differentiation challenging in experimental settings . In mice, BMP8B shares 74% amino acid identity with BMP8A within the mature region (aa 261-399) . While highly similar in structure, these proteins may have distinct spatial and temporal expression patterns during development and in adult tissues, necessitating carefully designed experiments to distinguish their individual contributions to biological processes.

What are the optimal techniques for detecting BMP8B in tissue samples?

For BMP8B detection in tissue samples, several validated techniques are available:

Western Blotting:

• Effective for detecting the 45 kDa pro-BMP8B form in tissue lysates

• Optimal conditions include using PVDF membranes with primary antibody concentration of 1 μg/mL

• Visualization with appropriate HRP-conjugated secondary antibodies (e.g., HAF016 for sheep primary antibodies)

Immunohistochemistry:

• Successfully detects BMP8B in paraffin-embedded tissue sections

• Recommended antibody concentration: 15 μg/mL with overnight incubation at 4°C

• Visualization using DAB-based detection systems with hematoxylin counterstaining

Researchers should optimize antibody concentrations for their specific tissue samples and include appropriate positive controls such as mouse brain tissue, where BMP8B expression has been confirmed .

How can I optimize Western blot protocols for BMP8B detection?

For optimal Western blot detection of BMP8B:

• Sample preparation:

  • Use reducing conditions to break disulfide bonds

  • Prepare tissue lysates using appropriate buffer systems (e.g., Immunoblot Buffer Group 8)

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for good resolution of the 45 kDa precursor form

  • Transfer to PVDF membrane, which has demonstrated superior results for BMP8B detection

• Antibody incubation:

  • Primary: Use 1 μg/mL of affinity-purified anti-BMP8B antibody

  • Secondary: HRP-conjugated secondary antibody appropriate for the primary antibody species

• Expected results:

  • Under reducing conditions, a specific band at approximately 45 kDa corresponding to pro-BMP8B

  • Mature BMP8B (~16 kDa) may be detected depending on tissue processing and antibody specificity

What are the established methods for measuring circulating BMP8B levels?

Enzyme-Linked Immunosorbent Assay (ELISA) is the established method for quantitative measurement of circulating BMP8B:

• Sample handling considerations:

  • Collect blood samples using standardized protocols

  • Process within 2 hours of collection

  • Store serum/plasma at -80°C to avoid degradation

• Assay parameters:

  • Research indicates a cut-off value of >51.59 pg/ml can discriminate between healthy controls and NASH patients

  • Reported sensitivity and specificity for NASH detection: 92.91% and 92.73%, respectively

• Clinical correlations:

  • BMP8B levels show positive correlations with liver function parameters including AST, ALT, APRI, and FIB-4 score in NASH patients

  • Negative correlation observed with HDL in NAFL patients

How is BMP8B implicated in nonalcoholic fatty liver disease progression?

Recent research has established a significant role for BMP8B in NAFLD pathogenesis:

• Expression pattern:

  • Circulatory BMP8B levels are elevated in NAFLD patients, with concentrations increasing with disease severity

  • Highest levels observed in NASH patients compared to simple steatosis (NAFL) and healthy controls

• Tissue expression:

  • Increased BMP8B mRNA expression in hepatic tissue of NASH patients

  • BMP8B expression increases parallel to fibrosis score in hepatic tissues

• Biomarker potential:

  • BMP8B maintained significant association with NASH in multivariate logistic regression analysis

  • High sensitivity (92.91%) and specificity (92.73%) for discriminating NASH patients from healthy controls

  • Cut-off value of >51.59 pg/ml effectively separates healthy controls from NASH patients

• Clinical correlations:

  • Significant positive correlation between BMP8B and key clinical parameters:

    • Aspartate aminotransferase (r = 0.31, p = 0.005)

    • Alanine aminotransferase (r = 0.23, p = 0.045)

    • APRI score (r = 0.30, p = 0.009)

    • FIB-4 score (r = 0.25, p = 0.036)

How does BMP8B compare to other non-invasive biomarkers for NAFLD/NASH?

When compared to established non-invasive biomarkers, BMP8B demonstrates promising diagnostic characteristics:

BMP8B offers several advantages over existing markers:

• More direct reflection of disease pathophysiology

• Higher combined sensitivity and specificity for NASH detection

• Potential for earlier disease detection based on mechanistic involvement

What is the tissue distribution of BMP8B in normal and pathological states?

BMP8B expression has been documented in several tissues:

• Normal states:

  • Brain tissue shows detectable BMP8B expression as confirmed by Western blot analysis

  • Reproductive tissues express BMP8B during development, with roles in germ cell lineage commitment

• Pathological states:

  • Liver: Significantly upregulated in NASH compared to healthy liver

  • Osteosarcoma: BMP8B has been detected in human osteosarcoma tissue by immunohistochemistry

  • Expression increases parallel to fibrosis score in NAFLD/NASH patients

How should I validate the specificity of BMP8B antibodies?

Rigorous validation of BMP8B antibodies is essential due to the high sequence homology with BMP8A (74% amino acid identity in mice):

• Cross-reactivity testing:

  • Test against recombinant BMP8A protein

  • Compare detection in tissues with known differential expression of BMP8A vs. BMP8B

  • Consider using BMP8A/B knockout models when available

• Western blot validation:

  • Confirm detection of the expected molecular weight band (45 kDa for pro-BMP8B)

  • Pre-absorption with immunizing peptide should eliminate specific staining

  • Multiple antibodies targeting different epitopes should yield consistent results

• Functional validation:

  • For neutralizing antibodies, confirm inhibition of BMP8B-specific signaling

  • Verify absence of effect on closely related BMP signaling pathways

What are the optimal storage and handling conditions for BMP8B antibodies?

To maintain antibody functionality and stability:

• Storage recommendations:

  • 12 months from date of receipt at -20 to -70°C as supplied

  • 1 month at 2-8°C under sterile conditions after reconstitution

  • 6 months at -20 to -70°C under sterile conditions after reconstitution

• Handling precautions:

  • Use a manual defrost freezer to store antibodies

  • Avoid repeated freeze-thaw cycles

  • Reconstitute only the amount needed for immediate use

  • Aliquot reconstituted antibody for long-term storage

How can I design experiments to study BMP8B signaling pathways?

When investigating BMP8B signaling mechanisms:

• Pathway components to examine:

  • Canonical signaling: Phosphorylation of SMAD1/5/8

  • Non-canonical pathways: p38 MAPK, ERK activation

  • Receptor complexes: Type I (ALK2, ALK3, ALK6) and Type II (BMPR2, ActR2A, ActR2B) receptors

• Experimental approaches:

  • Stimulation experiments with recombinant BMP8B protein

  • Loss-of-function studies using neutralizing antibodies or gene knockdown

  • Receptor blocking experiments to identify specific receptor utilization

  • Compare signaling kinetics and magnitude with other BMPs

• Readouts to evaluate:

  • Western blotting for phosphorylated SMADs

  • Reporter assays (BRE-luciferase) for transcriptional activity

  • qPCR for target gene induction (ID1, ID2, SMAD6, etc.)

  • Functional responses relevant to the tissue/cell type under investigation

How can I distinguish between BMP8A and BMP8B in experimental settings?

Differentiating between the highly homologous BMP8A and BMP8B requires specialized approaches:

• mRNA detection:

  • Design PCR primers targeting unique regions in the transcripts

  • Use highly specific TaqMan probes for qRT-PCR

  • Validate primer specificity using overexpression systems

• Protein detection:

  • Select antibodies raised against regions with sequence differences

  • Validate using recombinant proteins of both BMP8A and BMP8B

  • Consider mass spectrometry for definitive identification

• Functional studies:

  • Use isoform-specific siRNA knockdown

  • CRISPR-Cas9 gene editing targeting unique sequences

  • Rescue experiments with isoform-specific constructs

What are the key considerations for analyzing post-translational processing of BMP8B?

BMP8B undergoes complex post-translational modifications that affect detection and activity:

• Processing events to monitor:

  • Signal peptide removal (19 aa)

  • Proteolytic cleavage of prosegment (241 aa)

  • Dimerization via disulfide bond formation at Cys363

• Detection strategies:

  • Use antibodies recognizing different domains to distinguish processing forms

  • Run non-reduced vs. reduced samples to assess dimerization

  • Consider pulse-chase experiments to track processing kinetics

• Functional implications:

  • Prosegment removal is required for receptor binding and signaling

  • Dimerization is essential for biological activity

  • Processing efficiency may vary between tissues and pathological states

How should BMP8B research be integrated with broader studies of BMP signaling?

To position BMP8B research within the BMP field:

• Comparative analyses:

  • Side-by-side testing with other BMPs for signaling potency

  • Receptor utilization compared to related BMPs

  • Target gene profiles and signaling kinetics

• Antagonist interactions:

  • Sensitivity to extracellular antagonists (Noggin, Chordin, etc.)

  • Regulation by intracellular inhibitors (SMAD6/7)

  • Competition assays with other BMPs for receptor binding

• Systems biology approaches:

  • Network analysis positioning BMP8B within the BMP/TGF-β signaling network

  • Mathematical modeling of pathway dynamics

  • Multi-omics integration to understand context-dependent functions