ostf1 Antibody

What is OSTF1 and why is it significant in research?

OSTF1 is a small intracellular protein containing an SH3 domain followed by four ankyrin domains. It was first identified as a factor involved in the indirect activation of osteoclasts and bone-resorption activity in cell culture assays. OSTF1 has been linked to numerous conditions including spinal muscular atrophy through its interaction with SMN1, and is one of six genes deleted in a human developmental microdeletion syndrome at 9q21.13 . Knockout studies have demonstrated its role in bone development, with OSTF1 knockout mice showing increased trabecular mass in long bones . Its interactions with proteins such as F-actin, c-Src, and the E3 ubiquitin-protein ligase Cbl suggest diverse functional roles in cellular signaling and cytoskeletal organization .

What is the expression pattern of OSTF1 in mammalian tissues?

OSTF1 demonstrates widespread expression across multiple tissue types with notable tissue-specific patterns. Western blotting and X-Gal staining in knockout/reporter mice reveal OSTF1 expression in:

• Central Nervous System: Expressed at varying levels throughout the brain, with highest expression in neurons of the parabrachial nucleus. Also expressed in ventricles, choroid plexus, and oligodendrocytes .

• Vasculature: Strongly expressed in blood vessels of most organs .

• Spinal Cord: Present in motor neurons and nociceptors .

• Eyes: Found in ganglion cell layer, outer plexiform layer, retinal pigmented epithelium, and cornea .

• Bone Cells: Expressed in both osteoclasts and osteoblasts throughout differentiation .

This expression pattern suggests OSTF1 may play roles in multiple physiological processes beyond its initially identified function in bone metabolism.

What are the validated applications for OSTF1 antibodies?

Based on published research, OSTF1 antibodies have been successfully employed in:

• Western Blotting: Multiple antibodies have been validated, including rabbit anti-OSTF1 (Bethyl laboratories, A303-004A) at 1:10,000 dilution and rabbit anti-OSTF1 (Atlas HPA020514) at 1:200 dilution .

• Immunoprecipitation: Anti-OSTF1 antibodies can be used to isolate OSTF1 and its binding partners .

• ELISA: Solid-phase ELISA kits are available for quantitative determination of OSTF1 in various sample types including cell culture supernatant, plasma, serum, and tissue homogenates .

When selecting an OSTF1 antibody, researchers should consider the specific application, species reactivity, and epitope recognition to ensure optimal results.

What controls should be included when using OSTF1 antibodies?

Rigorous experimental design requires appropriate controls:

• Positive Controls: Tissues with known high OSTF1 expression (e.g., specific brain regions, vascular tissues).

• Negative Controls: Tissues from OSTF1 knockout models where protein should be undetectable by Western blotting, as confirmed in research using Ostf1^lacZ/lacZ mice .

• Loading Controls: Use of housekeeping proteins (e.g., β-actin as used in previous studies) for Western blotting .

• Secondary Antibody Controls: Omitting primary antibody to assess non-specific binding.

• Peptide Competition: Pre-incubation of antibody with immunizing peptide to confirm specificity.

These controls help validate antibody specificity and ensure reliable experimental outcomes.

How can OSTF1 antibodies be used to investigate protein-protein interactions?

OSTF1 interacts with multiple proteins including F-actin, c-Src, Cbl, SMN1, and SMN2. Researchers can employ several antibody-based approaches to study these interactions:

• Co-immunoprecipitation: Pull down OSTF1 using specific antibodies and identify binding partners through Western blotting or mass spectrometry. Previous research utilized V5-tagged OSTF1 in HEK293 cells followed by anti-V5 immunoprecipitation and LC-MS/MS protein identification .

• Proximity Ligation Assays: Visualize OSTF1 interactions with potential partners in situ at subcellular resolution.

• Western Blotting Following Fractionation: Determine subcellular localization of OSTF1 and co-localization with interaction partners.

What experimental approaches can elucidate OSTF1's role in bone development?

OSTF1 knockout mice demonstrate increased trabecular mass in long bones, confirming its role in bone development . Researchers can leverage antibodies to investigate mechanisms through:

• Immunohistochemistry/Immunofluorescence: Track OSTF1 expression and localization during bone development and remodeling.

• Cell-specific Expression Analysis: RT-PCR studies have confirmed Ostf1 mRNA in both differentiating osteoclasts (from day 0 to fully differentiated day 5) and throughout osteoblast differentiation .

• Quantitative Analysis: Use ELISA to measure OSTF1 levels in bone cells under various conditions or treatments.

• Micro-CT Analysis: Combine with immunohistochemistry to correlate OSTF1 expression with bone microarchitecture parameters as demonstrated in OSTF1 knockout studies .

• Functional Assays: Use neutralizing antibodies to block OSTF1 function in bone cell cultures and assess effects on differentiation, activity, and signaling.

How can researchers address challenges in detecting OSTF1 in tissues with low expression?

The search results indicate that OSTF1 is widely expressed but at varying levels, with some regions showing lower expression . To overcome detection challenges:

• Signal Amplification: Use tyramide signal amplification or similar techniques for immunohistochemistry/immunofluorescence applications.

• Sensitive Detection Systems: Employ enhanced chemiluminescence or fluorescence-based detection for Western blotting.

• Sample Enrichment: Consider subcellular fractionation to concentrate OSTF1 from tissues with low expression.

• Reporter Systems: Utilize reporter systems like the LacZ knockin approach described in the search results, which allows visualization of expression at the single-cell level .

• Complementary Techniques: Combine protein detection with mRNA analysis (RT-PCR) to validate expression, as demonstrated for osteoclast and osteoblast expression .

What are common issues with OSTF1 antibody specificity and how can they be addressed?

Ensuring antibody specificity is crucial for reliable results. Common challenges include:

• Cross-reactivity: Validate using tissues from OSTF1 knockout models, as demonstrated in the research where OSTF1 protein was undetectable by Western blotting in Ostf1^lacZ/lacZ tissues .

• Inconsistent Results Between Applications: An antibody may work well for Western blotting but poorly for immunohistochemistry. Optimize protocols for each application separately.

• Batch-to-batch Variation: Use consistent lots when possible and revalidate with new batches.

• Epitope Masking: If post-translational modifications or protein interactions mask epitopes, try multiple antibodies targeting different regions of OSTF1.

What factors affect the success of OSTF1 ELISA assays?

When using OSTF1 ELISA kits for quantitative analysis, researchers should consider:

• Sample Preparation: Ensure proper processing of cell culture supernatant, plasma, serum, or tissue homogenates .

• Washing Technique: The search results specifically note that "Incomplete washing will adversely affect the test outcome" .

• Standard Curve Generation: Prepare accurate dilutions and ensure proper curve fitting.

• Assay Validation: Include known positive and negative controls.

• Sample Dilution: Optimize sample dilution to ensure measurements fall within the linear range of the assay.

How might OSTF1 antibodies contribute to understanding neurodevelopmental disorders?

Given OSTF1's link to spinal muscular atrophy through SMN1 interaction and its deletion in a developmental microdeletion syndrome , antibody-based approaches could:

• Map Developmental Expression: Use immunohistochemistry to characterize OSTF1 expression during neural development.

• Investigate Protein Interactions: Explore OSTF1-SMN1 interactions in neural tissues using co-immunoprecipitation and proximity ligation assays.

• Comparative Studies: Analyze OSTF1 expression in animal models of neurodevelopmental disorders versus controls.

• Functional Studies: Use antibodies to block or detect OSTF1 in neuronal cultures to assess effects on neuronal development and function.

What is the potential for using OSTF1 antibodies in vascular research?

The widespread expression of OSTF1 in the vasculature of most organs suggests potential roles in vascular biology. Researchers might:

• Create Vascular Expression Maps: Use immunohistochemistry to characterize OSTF1 expression across different vascular beds.

• Investigate Angiogenesis: Study OSTF1 expression during vascular development and angiogenesis.

• Explore Disease Models: Examine OSTF1 expression in models of vascular diseases, potentially relevant to its association with coronary artery diseases .

• Cell-specific Analysis: Determine which vascular cell types (endothelial cells, smooth muscle cells, pericytes) express OSTF1 and under what conditions.