BMP-7 Antibody

What is BMP-7 and what cellular functions does it regulate?

Bone Morphogenetic Protein 7 (BMP7) belongs to the transforming growth factor-beta (TGF-β) superfamily. It functions as a secreted signaling molecule involved in multiple developmental and physiological processes. BMP7 plays crucial roles in bone formation and morphogenesis, but its functions extend well beyond skeletal development. Research has demonstrated that BMP7 promotes the differentiation of Langerhans cells in the epidermis during prenatal development through the activation of the ALK3-Smad1/5/8 signaling pathway . BMP7 has a calculated molecular weight of 49 kDa, though it is typically observed at 43-49 kDa in experimental conditions . The protein is encoded by the BMP7 gene (Gene ID: 655, UNIPROT ID: P18075) and exerts its biological effects through specific receptor binding and downstream signaling cascades .

How do I select the appropriate BMP-7 antibody for my experimental applications?

Selecting the appropriate BMP-7 antibody requires consideration of several critical factors:

• Application compatibility: Verify that the antibody has been validated for your intended application. For example, antibody 12221-1-AP has been validated for Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), and ELISA applications .

• Species reactivity: Confirm that the antibody recognizes BMP-7 in your species of interest. For instance, 12221-1-AP shows reactivity with human samples and has cited reactivity with rat samples .

• Antibody format: Consider whether a polyclonal or monoclonal antibody best suits your experimental needs. Polyclonal antibodies like 12221-1-AP provide broader epitope recognition, while monoclonal antibodies like clone 164311 offer higher specificity for a single epitope .

• Validation data: Review the antibody's validation data to ensure it performs reliably in your specific application. Published literature citing the antibody provides valuable information about real-world performance.

What are the recommended dilution ratios for different BMP-7 antibody applications?

The optimal dilution ratios for BMP-7 antibodies vary by application and specific antibody. For the widely used BMP-7 antibody 12221-1-AP, the following dilutions are recommended:

It is important to note that these ratios should be optimized for each specific experimental system. The manufacturer recommends that "this reagent should be titrated in each testing system to obtain optimal results" as the optimal dilution may be sample-dependent .

How should I optimize Western blot protocols for BMP-7 detection?

For optimal Western blot detection of BMP-7, follow these methodological guidelines:

• Sample preparation: Extract proteins using standard lysis buffers containing protease inhibitors to prevent degradation of BMP-7.

• Protein loading: Load 20-50 μg of total protein per lane, particularly for detecting endogenous BMP-7.

• Separation: Use 10-12% SDS-PAGE gels for optimal separation of BMP-7, which has an observed molecular weight of 43-49 kDa .

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard protocols.

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute BMP-7 antibody 1:500-1:2000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash 3-5 times with TBST to remove unbound antibody.

• Secondary antibody: Use the appropriate HRP-conjugated secondary antibody based on the primary antibody host species (e.g., anti-rabbit for 12221-1-AP).

• Detection: Visualize using ECL or similar detection systems.

For positive controls, MCF-7 and HEK-293 cells have been validated for BMP-7 expression and can be used as reference standards .

What is the recommended protocol for immunohistochemical detection of BMP-7?

For immunohistochemical detection of BMP-7 in tissue samples:

• Tissue preparation: Fix tissues in formalin and embed in paraffin following standard protocols.

• Sectioning: Cut sections at 4-6 μm thickness and mount on positively charged slides.

• Deparaffinization: Remove paraffin with xylene and rehydrate through graded alcohols.

• Antigen retrieval: For BMP-7 antibody 12221-1-AP, use TE buffer pH 9.0 for optimal antigen retrieval. Alternatively, citrate buffer pH 6.0 can be used .

• Endogenous peroxidase block: Quench endogenous peroxidase activity with 3% hydrogen peroxide.

• Protein block: Apply protein blocking solution to reduce non-specific binding.

• Primary antibody: Dilute BMP-7 antibody to 1:50-1:500 and incubate sections according to optimized conditions (typically overnight at 4°C) .

• Detection system: Apply appropriate detection system based on primary antibody host species.

• Chromogen development: Develop with DAB or other suitable chromogen.

• Counterstain: Counterstain with hematoxylin, dehydrate, and mount.

Human kidney tissue, bladder tissue, and renal cell carcinoma tissue have been validated as positive controls for BMP-7 immunohistochemical staining .

How can I detect BMP-7 autoantibodies in research subjects?

For detecting BMP-7 autoantibodies (BMP7-aAB) in serum samples, researchers have established a specialized protocol:

• Sample preparation: Use 10 μl of serum per reaction and incubate with diluted MACN-labelled rhBMP7 (100 μl per reaction) overnight at 4°C .

• Antibody capture: Incubate with 50 μl of 10% protein A slurry in PBS for 1 hour at room temperature with shaking at 300 rpm to bind IgG antibodies .

• Washing step: Wash the protein A pellet containing the labelled BMP-bound IgG three times with washing buffer .

• Detection: Measure chemiluminescence, which directly correlates to the amount of BMP7-aAB present in the sample .

• Data analysis: Classify samples as positive when measured signals exceed a floating cut point calculated by adding 1.5 times the interquartile range (IQR) to the value defining the 75th percentile .

This assay has been validated to detect autoantibodies over almost two orders of magnitude concentration (0.4-25 μg/ml) and can be used to monitor autoantibody development in response to rhBMP7 treatment .

How does BMP-7 function in immunomodulation and cancer therapy resistance?

Recent research has revealed significant roles for BMP-7 in immunomodulation and cancer therapy resistance:

• Immunotherapy resistance: Overexpression of BMP7 has been identified as a mechanism for resistance to anti-PD1 therapy in preclinical models and in patients with disease progression while on immunotherapies .

• Immune cell modulation: BMP7 secreted by tumor cells acts on macrophages and CD4+ T cells in the tumor microenvironment, inhibiting MAPK14 expression and impairing pro-inflammatory responses .

• Therapeutic targeting: Knockdown of BMP7 or its neutralization via follistatin in combination with anti-PD1 therapy re-sensitizes resistant tumors to immunotherapies .

These findings identify the BMP7 signaling pathway as a potential immunotherapeutic target in cancer treatment. Researchers investigating this phenomenon should consider experimental designs that:

• Assess BMP-7 expression levels in tumor samples before and after immunotherapy

• Evaluate immune cell phenotypes and functions in the presence of BMP-7

• Test combinations of BMP-7 inhibition and immunotherapy agents

• Monitor MAPK14 expression as a downstream readout of BMP-7 activity

What is the relationship between BMP-7 and TGF-β1 in cellular differentiation?

BMP-7 and TGF-β1 exhibit distinct and sometimes opposing effects on cellular differentiation, particularly in the context of Langerhans cell development:

• Receptor specificity: BMP-7 preferentially signals through ALK3 (BMPR-IA), while TGF-β1 signals through ALK5 (TGF-βRI) .

• Signaling pathways: BMP-7 activates the Smad1/5/8 pathway, whereas TGF-β1 activates the Smad2/3 pathway .

• Functional outcomes: Research has demonstrated that BMP7-dependent activation of ALK3-Smad1/5/8 promotes Langerhans cell differentiation more effectively than TGF-β1 .

• Proliferative effects: BMP-7 induces superior proliferation compared to TGF-β1-treated cells, as confirmed by CFSE-labeling experiments .

• Cluster formation: BMP-7 alone induces higher frequencies of Langerhans cell clusters than TGF-β1 .

These findings suggest that BMP-7 acts as a TGF-β1-independent positive regulator of Langerhans cell differentiation, and the selective triggering of ALK3 by BMP-7 enables amplified Langerhans cell generation . Researchers studying differentiation processes should consider these distinct signaling pathways when designing experiments and interpreting results.

How do BMP-7-induced autoantibodies affect experimental and clinical outcomes?

The development of autoantibodies against BMP-7 (BMP7-aAB) following rhBMP7 treatment has important implications for both experimental research and clinical applications:

• Prevalence: The baseline prevalence of BMP7-aAB is 1-2.5% in non-treated patients or healthy controls. Following rhBMP7 treatment, up to 18% of patients develop detectable BMP7-aAB at four weeks post-treatment .

• Functional activity: IgG from BMP7-aAB positive sera inhibits BMP7-reporter gene activity in a dose-dependent manner in vitro, demonstrating functional antagonism of BMP7 signaling .

• Transient nature: In patients treated with rhBMP7, antibody levels typically return to non-detectable levels within six months, indicating the transient nature of this immune response .

• Clinical impact: Despite the development of neutralizing antibodies, successful consolidation of bone fractures was observed in the majority of both aAB-positive and aAB-negative patients, suggesting that the presence of these antibodies does not necessarily preclude treatment success .

These findings have important methodological implications for researchers working with rhBMP7:

• Pre-screening for natural autoantibodies may be advisable in certain experimental contexts

• Monitoring antibody development during treatment periods can help interpret variable responses

• The transient nature of the antibody response should be considered when designing longitudinal studies

• The potential functional antagonism of BMP7 signaling by autoantibodies should be accounted for when interpreting experimental outcomes

What are common challenges in immunoprecipitation of BMP-7 and how can they be addressed?

Immunoprecipitation (IP) of BMP-7 presents several technical challenges that researchers should be prepared to address:

• Antibody selection: Choose antibodies specifically validated for IP applications. For BMP-7 antibody 12221-1-AP, use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Protein abundance: BMP-7 may be expressed at low levels in some cellular contexts, making detection challenging. HEK-293 cells have been validated as a positive control system for BMP-7 IP .

• Protein-protein interactions: BMP-7 interacts with numerous proteins that may affect antibody binding. Consider using mild lysis conditions to preserve these interactions if they are of interest.

• Non-specific binding: Pre-clear lysates with protein A/G beads before adding the BMP-7 antibody to reduce background.

• Signal detection: After IP, use Western blotting with another BMP-7 antibody (if possible) to confirm specificity of the pulled-down protein. The expected molecular weight range is 43-49 kDa .

How can researchers distinguish between autoantibodies to BMP-7 and other BMP family members?

Distinguishing between autoantibodies to different BMP family members requires careful assay design and validation:

• Specific antigen labeling: Use individually MACN-labelled rhBMP proteins (e.g., rhBMP2, rhBMP7) as bait for autoantibodies in parallel assays .

• Cross-reactivity assessment: Test serum samples with multiple BMP family members to identify cross-reactive antibodies. Research has shown that the number of samples with cross-reaction towards both BMP7 and BMP2 is relatively small (n = 4 in one study) .

• Competitive binding: Perform competitive binding assays with unlabeled BMPs to confirm specificity.

• Signal quantification: Use standardized cutoff values to classify samples as positive for specific BMP autoantibodies. One validated approach defines positivity when measured signals exceed a floating cut point calculated by adding 1.5 times the interquartile range (IQR) to the value defining the 75th percentile .

• Control samples: Include known positive and negative controls in each assay. Mouse monoclonal antibodies against specific BMPs can serve as positive controls .

What factors affect the reproducibility of BMP-7 antibody experiments?

Several factors can impact the reproducibility of experiments using BMP-7 antibodies:

• Antibody quality and lot-to-lot variation: Different lots of the same antibody may have varying performance characteristics. Document lot numbers and maintain consistent sourcing.

• Sample preparation: Variations in protein extraction methods, buffer composition, and protein quantification can affect results. Standardize these protocols across experiments.

• Storage conditions: BMP-7 antibodies should be stored according to manufacturer recommendations. For antibody 12221-1-AP, store at -20°C in PBS with 0.02% sodium azide and 50% glycerol pH 7.3 .

• Antigen retrieval methods: For IHC applications, consistent antigen retrieval is critical. For BMP-7 antibody 12221-1-AP, TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 is an alternative .

• Detection systems: Variations in secondary antibodies and detection reagents can affect signal intensity. Standardize these components across experiments.

• Positive controls: Include validated positive controls in each experiment. For Western blotting, MCF-7 and HEK-293 cells have been validated for BMP-7 expression .

How is BMP-7 being investigated as a therapeutic target in cancer immunotherapy?

Recent research has identified BMP-7 as a potential therapeutic target in cancer immunotherapy:

• Resistance mechanism: Overexpression of BMP7 represents a mechanism for resistance to anti-PD1 therapy in preclinical models and in patients with progressive disease on immunotherapies .

• Cellular mechanisms: BMP7 secreted by tumor cells acts on macrophages and CD4+ T cells in the tumor microenvironment, inhibiting MAPK14 expression and impairing pro-inflammatory responses essential for effective immunotherapy .

• Therapeutic approach: Knockdown of BMP7 or its neutralization via follistatin in combination with anti-PD1 therapy re-sensitizes resistant tumors to immunotherapies, suggesting a potential combination approach for overcoming resistance .

These findings highlight the BMP7 signaling pathway as a promising immunotherapeutic target. Current research is focused on developing specific BMP-7 inhibitors or neutralizing antibodies that could be combined with existing immunotherapies to improve patient outcomes.

What are the latest findings regarding BMP-7's role in cellular differentiation beyond bone formation?

Beyond its well-established role in bone formation, recent research has expanded our understanding of BMP-7's functions in cellular differentiation:

• Langerhans cell development: BMP7 promotes the differentiation of Langerhans cells in the epidermis during prenatal development through ALK3-Smad1/5/8 signaling .

• Comparative efficacy: BMP7 far exceeds TGF-β1 in promoting Langerhans cell generation, inducing higher frequencies of LC clusters and enhanced proliferation .

• Mechanistic independence: BMP7 acts as a TGF-β1-independent positive regulator of LC differentiation, operating through distinct signaling pathways .

These findings suggest that BMP-7 plays crucial roles in immune cell development and differentiation beyond its classical functions in bone and kidney development. The superior efficacy of BMP-7 in promoting certain differentiation processes compared to other TGF-β family members highlights its potential utility in directed differentiation protocols for research and therapeutic applications.