BRINP3 Antibody

What is BRINP3 and what cellular functions does it perform?

BRINP3 (BMP/retinoic acid inducible neural specific 3) is a member of the BRINP protein family with a canonical length of 766 amino acid residues and a molecular mass of approximately 88.4 kDa in humans . Functionally, BRINP3 inhibits neuronal cell proliferation through negative regulation of cell cycle transition processes . The protein has been identified in multiple cellular compartments, primarily in mitochondria, and is also known to be secreted extracellularly .

Recent research has revealed that BRINP3 plays a significant role in tumor biology. It functions as an oncogene in osteosarcoma, where its high expression (observed in 64.13% of human osteosarcoma tissues) correlates with histological grade, tumor recurrence, and poor clinical prognosis . Mechanistically, BRINP3 has been shown to interact with microtubule-associated protein 4 (MAP4) at the protein level, with this interaction potentially mediating its effects on cell proliferation and invasion in osteosarcoma cells .

What are the common applications for BRINP3 antibodies in research?

BRINP3 antibodies are employed in multiple experimental techniques, with Western blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) being the most commonly utilized applications . These antibodies are critical for the immunodetection of BRINP3 protein in various research contexts.

Immunohistochemistry (IHC) represents another significant application, particularly in clinical and pathological studies. In osteosarcoma research, anti-BRINP3 antibodies have been used for tumor tissue staining at dilutions of 1:400, followed by incubation with secondary antibody kits and digital scanning for quantitative analysis . The staining results are typically scored based on both intensity (0-3 scale) and positive area percentage (0-4 scale), with combined scores determining expression levels .

For cellular and molecular studies, BRINP3 antibodies are employed in immunoprecipitation assays to investigate protein-protein interactions, as demonstrated in studies identifying BRINP3's interaction with MAP4, EIF3F, and PARP1 .

What is the subcellular localization pattern of BRINP3?

BRINP3 exhibits a complex subcellular distribution pattern that includes mitochondrial localization and extracellular secretion . More detailed examination has revealed that BRINP3 can also be found in dendrites, the endoplasmic reticulum, and the extracellular region . This multifaceted localization pattern suggests that BRINP3 likely performs different functions depending on its cellular context and compartmentalization.

The dual nature of BRINP3 as both an intracellular and secreted protein makes it particularly interesting from a research perspective, as it may participate in both autocrine/paracrine signaling and intracellular regulatory processes. When designing experiments to study BRINP3, researchers should consider this dual localization and select appropriate fractionation and detection methods that can effectively capture both pools of the protein.

How does BRINP3 expression vary between normal and pathological tissues?

BRINP3 demonstrates significant expression variation across normal and pathological tissues. In normal tissues, BRINP3 is highly expressed in oral keratinocytes, contrasting with its weak expression in tongue squamous cell carcinoma (SCC) . This downregulation in tongue SCC suggests a potential tumor suppressor role in this specific cancer type.

Conversely, in osteosarcoma, BRINP3 displays markedly elevated expression, with 64.13% of human osteosarcoma tissues showing high BRINP3 levels . This upregulation correlates significantly with clinical parameters including histological grade, tumor recurrence, and poor prognosis in osteosarcoma patients . The differential expression patterns across cancer types highlight the context-dependent nature of BRINP3's function in various tissues.

Additionally, BRINP3's involvement in neurological contexts is significant, with dysregulation implicated in several neurodegenerative conditions including Alzheimer's disease, Parkinson's disease, and schizophrenia . This tissue-specific and disease-dependent expression pattern makes BRINP3 a complex but valuable target for research across multiple fields.

What validation methods should be used to confirm BRINP3 antibody specificity?

Establishing antibody specificity is crucial for generating reliable BRINP3 research data. A comprehensive validation approach should include:

• Western blot analysis using both recombinant BRINP3 protein and endogenous BRINP3 from tissues known to express the protein (such as brain tissue or U-251MG cells) . The antibody should detect a band at the expected molecular weight of approximately 88.4 kDa .

• Peptide competition assays where pre-incubation of the antibody with the immunizing peptide (such as the recombinant fusion protein containing amino acids 240-500 of human BRINP3) should eliminate or significantly reduce signal detection.

• Knockdown validation using BRINP3-specific shRNA in cells with endogenous BRINP3 expression. Researchers have successfully used lentivirus-based shRNA systems to reduce BRINP3 expression in osteosarcoma cell lines (U2OS and Saos-2), with validation by both qRT-PCR and western blotting .

• Cross-reactivity testing across species, as many BRINP3 antibodies show reactivity with human, mouse, and rat samples , allowing for comparative studies across model organisms.

What methodological considerations are important when studying BRINP3-protein interactions?

When investigating BRINP3 protein interactions, researchers should implement a systematic approach combining multiple complementary techniques:

• Co-immunoprecipitation optimized for BRINP3: Effective protocols involve transfecting cells with FLAG-tagged BRINP3 constructs, followed by cell lysis using IP lysis buffer supplemented with protease inhibitors. After centrifugation, the supernatant should be incubated with appropriate antibodies and protein A beads for approximately 4 hours at 4°C. After washing, the precipitated proteins can be eluted and analyzed by western blotting .

• Mass spectrometry analysis: This approach has successfully identified BRINP3-interacting proteins such as MAP4, EIF3F, and PARP1 . Selection criteria should include unique peptide count ≥1 and peptide FDR <0.01 for reliable interaction identification.

• Verification of interactions: Following initial identification by mass spectrometry, interactions should be verified through reciprocal co-immunoprecipitation, where both BRINP3 and the interacting protein can pull down each other.

• Functional validation: To establish biological relevance, researchers should examine how overexpression or knockdown of the interacting protein (e.g., MAP4) affects BRINP3-mediated cellular processes. For example, studies have shown that MAP4 overexpression can partially reverse the inhibitory effects of BRINP3 knockdown on osteosarcoma cell proliferation and invasion .

How can researchers effectively establish and validate BRINP3 knockdown models?

Establishing reliable BRINP3 knockdown models requires careful consideration of several key factors:

• Optimal vector selection: For stable knockdown, lentiviral vectors such as pGV115-BRINP3-shRNA have proven effective . The vector selection should consider the inclusion of appropriate selection markers and reporter genes (such as GFP) to monitor transfection efficiency.

• Transfection protocol optimization: For lentiviral transduction of osteosarcoma cell lines, polybrene (8 µg/ml) has been used to enhance transduction efficiency . Researchers should aim for transfection efficiency exceeding 80% before proceeding with subsequent experiments.

• Knockdown validation at multiple levels:

  • mRNA level: Using qRT-PCR with SYBR Green chemistry and calculating relative expression by the 2^-ΔΔCT method with GAPDH as reference .

  • Protein level: Western blotting with validated BRINP3 antibodies, quantifying relative protein levels with GAPDH (1:5000) as loading control .

• Functional validation: Verify the biological impact of BRINP3 knockdown through appropriate functional assays. For osteosarcoma cells, both Celigo cell counting and MTT assays conducted over 5 days have successfully demonstrated reduced proliferation in BRINP3-knockdown cells . Similarly, transwell assays have confirmed decreased invasive activity following BRINP3 knockdown .

What are the optimal protocols for BRINP3 detection in tissue samples via immunohistochemistry?

For reliable BRINP3 detection in tissue samples via immunohistochemistry, researchers should follow these methodological guidelines:

• Sample preparation: Formalin-fixed, paraffin-embedded tissues should be sectioned at 4 µm thickness . Prior to staining, sections should be deparaffinized according to standard protocols.

• Antigen retrieval: Optimal retrieval has been achieved using sodium citrate buffer (pH 6.0) at high temperature and pressure for 2 minutes . This step is critical for exposing epitopes that may be masked during fixation.

• Antibody selection and dilution: Anti-BRINP3 antibodies (such as those from Signalway Antibody) have been successfully used at 1:400 dilution . Primary antibody incubation should be performed overnight at 4°C to ensure specific binding.

• Detection system: Following primary antibody incubation, sections should be treated with appropriate anti-mouse/rabbit IHC secondary antibody kits (such as those from Gene Tech) . Digital pathology slide scanners enable systematic analysis of stained sections.

• Scoring system implementation: BRINP3 expression should be evaluated using a standardized scoring system that incorporates both staining intensity (0-3 scale) and positive area percentage (0-5%, 5-25%, 26-50%, 51-75%, ≥75%) . Final scores can be calculated by adding these two parameters, with scores ≥3 typically defined as high BRINP3 expression .

How do different experimental conditions affect BRINP3 antibody performance in Western blot applications?

The performance of BRINP3 antibodies in Western blot applications can be significantly influenced by experimental conditions:

• Sample preparation considerations:

  • BRINP3 is localized in multiple cellular compartments including mitochondria and is secreted . Therefore, complete protein extraction requires lysis buffers containing appropriate detergents.

  • RIPA lysis buffer containing 1% phosphatase and protease inhibitors has proven effective for BRINP3 extraction .

  • Protein quantification using BCA protein assay kits ensures consistent loading across samples .

• Optimization of antibody dilution:

  • Different BRINP3 antibodies require specific dilution ranges. For example, rabbit polyclonal BRINP3 antibodies have been successfully used at dilutions between 1:500 and 1:2000 for Western blot applications .

  • Titration experiments should be performed to determine the optimal concentration for each specific antibody.

• Membrane selection and blocking parameters:

  • PVDF membranes (Millipore) have been successfully used for BRINP3 detection .

  • Blocking with 5% fat-free milk at room temperature for 1 hour provides adequate blocking while maintaining antibody binding capacity .

• Incubation conditions:

  • Primary antibody incubation at 4°C overnight followed by secondary antibody (1:2000) incubation for 1 hour at room temperature has yielded optimal results .

  • Multiple washing steps with 1× TBST between antibody incubations are essential for reducing background signal.

• Detection method selection:

  • Enhanced chemiluminescence (ECL) reagents have successfully visualized BRINP3 bands, with GAPDH (1:5000) serving as an effective loading control .

What approaches can be used to study the role of BRINP3 in neurodegenerative disease models?

Investigating BRINP3's role in neurodegenerative diseases requires specialized experimental approaches:

• Expression analysis in disease models:

  • Quantitative comparison of BRINP3 expression in brain tissues from patients with Alzheimer's disease, Parkinson's disease, and schizophrenia versus age-matched controls .

  • Analysis across different brain regions to identify region-specific alterations.

• Cellular models:

  • Establishment of BRINP3 overexpression and knockdown in neuronal cell lines using lentiviral vectors similar to those employed in osteosarcoma studies .

  • Evaluation of neuronal differentiation, survival, and morphology following BRINP3 modulation.

• Functional assessment:

  • Analysis of cell cycle parameters, as BRINP3 is known to inhibit neuronal cell proliferation by negative regulation of cell cycle transition .

  • Apoptosis assays to determine BRINP3's impact on neuronal survival.

  • Axonal growth and dendrite formation assays, given BRINP3's localization in dendrites .

• Protein interaction studies:

  • Investigation of potential interactions between BRINP3 and proteins implicated in neurodegenerative diseases through co-immunoprecipitation followed by mass spectrometry.

  • Verification of interactions using appropriate antibodies against candidate interacting proteins.

• Animal models:

  • Development of transgenic mouse models with conditional BRINP3 knockout or overexpression in specific neuronal populations.

  • Behavioral testing to assess cognitive function, motor coordination, and other relevant parameters.

  • Immunohistochemical analysis of brain sections using optimized BRINP3 antibody protocols.

How can researchers distinguish between BRINP3 isoforms using antibody-based methods?

BRINP3 exists in up to two different isoforms , making isoform discrimination an important consideration in research. Effective discrimination requires:

• Epitope-specific antibody selection: Researchers should select antibodies raised against regions that differ between isoforms. For instance, antibodies targeting the recombinant fusion protein containing amino acids 240-500 of human BRINP3 (NP_950252.1) may detect specific isoforms depending on sequence conservation.

• Electrophoretic resolution: Using gradient SDS-PAGE gels (e.g., 4-15%) can improve separation of closely sized isoforms. Extended running times at lower voltages may further enhance resolution.

• Isoform-specific knockdown: Using siRNAs or shRNAs targeting unique regions of specific isoforms, followed by Western blot analysis, can help determine which bands correspond to which isoforms.

• Recombinant protein controls: Including recombinant proteins representing each isoform as positive controls in Western blots provides reference markers for band identification.

• Mass spectrometry validation: For definitive isoform identification, immunoprecipitation followed by mass spectrometry analysis can identify peptides unique to specific isoforms.

What control experiments are essential when evaluating BRINP3's role in cell cycle regulation?

When investigating BRINP3's reported function in cell cycle regulation , several control experiments are critical:

• Expression confirmation controls:

  • Verification of BRINP3 knockdown or overexpression at both mRNA level (via qRT-PCR) and protein level (via Western blot) .

  • Inclusion of multiple shRNA sequences or rescue experiments to confirm specificity of observed effects.

• Cell cycle analysis controls:

  • Positive controls: Treatment with known cell cycle inhibitors (e.g., nocodazole for G2/M arrest).

  • Negative controls: Non-targeting shRNA or empty vector controls.

  • Analysis of multiple cell cycle markers: Beyond proliferation assays, include specific markers for different cell cycle phases.

• Mechanism validation controls:

  • Analysis of known cell cycle regulators: Assess changes in cyclins, CDKs, and CKIs following BRINP3 modulation.

  • Pathway inhibition: Use specific inhibitors of pathways potentially affected by BRINP3 to confirm mechanistic relationships.

• Cell type specificity controls:

  • Perform experiments in multiple cell types, as BRINP3's expression varies between tissues .

  • Compare effects in normal versus cancer cells, considering BRINP3's differential expression in these contexts.

• Interaction verification:

  • For protein interactions potentially mediating BRINP3's effects on cell cycle (e.g., MAP4), include reverse immunoprecipitation and competition experiments .