OLFM2 Antibody

What is OLFM2 and what are its primary biological functions?

OLFM2 (Olfactomedin 2) is a secretory glycoprotein belonging to the family of olfactomedin domain-containing proteins. It has been identified as a novel regulator mediating smooth muscle differentiation through the TGF-β signaling pathway . OLFM2 expression increases markedly upon TGF-β treatment, with an 8.4-fold increase observed after 48 hours of treatment .

This protein plays significant roles in multiple physiological processes. In smooth muscle differentiation, OLFM2 functions by empowering serum response factor (SRF) binding to the CArG box in smooth muscle gene promoters, thereby promoting the expression of smooth muscle markers including SM α-actin, SM22α, and SM myosin heavy chain . In neurological systems, OLFM2 interacts with the GluR2 subunit of AMPA receptor complexes and is involved in the organization of these receptor complexes . Studies with OLFM2-deficient mice have demonstrated its importance in neurological functions, as these mice exhibit reduced exploration, locomotion, olfactory sensitivity, abnormal motor coordination, and anxiety-related behavior .

What tissue types express OLFM2 and where is it primarily localized?

OLFM2 expression has been detected in multiple tissue types. In the brain, OLFM2 is mainly expressed in the olfactory bulb, cortex, piriform cortex, olfactory trabeculae, and inferior and superior colliculus . This expression pattern has been demonstrated through β-galactosidase staining in OLFM2-deficient mice where the OLFM2 gene was replaced with the LacZ gene .

In the eye, OLFM2 expression is detected primarily in retinal ganglion cells . Western blot analysis has confirmed the presence of OLFM2 in human brain tissue, fetal human brain tissue, mouse brain tissue, and rat brain tissue . Additionally, OLFM2 expression has been observed in smooth muscle of human aorta tissue, supporting its role in vascular development .

At the subcellular level, OLFM2 has been found in both the cytoplasm and nuclei of cells. Interestingly, TGF-β stimulation causes the majority of OLFM2 to accumulate in the nuclei, suggesting its function as a nuclear factor in transcriptional regulation . Fractionation studies have shown that before TGF-β treatment, only 38% of OLFM2 is located in the nuclei, while after TGF-β induction, nuclear OLFM2 increases to 81% of the total protein .

What are the recommended applications for OLFM2 antibody?

Based on available commercial information, OLFM2 antibodies are primarily recommended for Western Blot (WB) and ELISA applications . For Western blot applications, the recommended dilution ranges from 1:500 to 1:3000, with some suppliers suggesting a range of 1:500 to 1:2000 . It is important to note that optimal dilutions should be determined by the researcher for each specific testing system to obtain optimal results .

The commercially available OLFM2 antibodies show reactivity with human, mouse, and rat samples . Positive Western blot detection has been specifically confirmed in human brain tissue, fetal human brain tissue, mouse brain tissue, and rat brain tissue . These antibodies are typically rabbit polyclonal antibodies generated against OLFM2 fusion proteins or specific OLFM2 immunogens .

For most effective application, researchers should consider the observed molecular weight of OLFM2, which ranges from 46-51 kDa, with a calculated molecular weight of 51 kDa based on its 454 amino acid sequence .

How can I optimize immunoprecipitation protocols to study OLFM2 protein interactions?

Optimizing immunoprecipitation (IP) protocols for OLFM2 requires careful consideration of protein interactions and complex formation. Based on published research, OLFM2 physically interacts with serum response factor (SRF) and forms part of protein complexes with AMPA receptors . For effective IP experiments, consider the following methodological approach:

• Sample preparation: For studying OLFM2-SRF interactions, extract proteins from cells treated with and without TGF-β, as this treatment enhances their interaction . For neurological studies, prepare synaptosomal membrane fractions from brain cortex or retina tissue where OLFM2 co-segregates with AMPA receptor complexes .

• Lysis conditions: Use buffers that preserve protein-protein interactions while effectively solubilizing membrane proteins. For OLFM2-SRF interactions, standard Co-IP buffers are effective as demonstrated in previous studies . For AMPA receptor complex interactions, include mild detergents that maintain the integrity of receptor complexes .

• Antibody selection: The choice between anti-OLFM2 or antibodies against binding partners (like anti-SRF or anti-GluR2) depends on the specific question. Previous studies have successfully used both approaches - anti-OLFM2 antibodies to pull down SRF and anti-SRF antibodies to pull down OLFM2 .

• Controls: Include appropriate negative controls such as IgG from the same species as the primary antibody, and positive controls using known interaction partners. For OLFM2 studies, samples from OLFM2 knockout mice or cells with OLFM2 knockdown provide excellent negative controls .

When analyzing results, note that TGF-β treatment enhances OLFM2-SRF interaction, likely due to increased expression of OLFM2 induced by TGF-β . In neurological studies, immunoprecipitation from synaptosomal membrane fractions of OLFM2 null mouse brain cortex using GluR2 antibody showed reduced levels of several components of the AMPAR complex, suggesting OLFM2's role in organizing these receptor complexes .

What are the critical considerations when using OLFM2 antibody in chromatin immunoprecipitation (ChIP) assays?

Chromatin immunoprecipitation (ChIP) assays have proven valuable in understanding OLFM2's role in transcriptional regulation, particularly in studying how OLFM2 affects SRF binding to CArG boxes in smooth muscle gene promoters. Based on published research, consider these critical factors when designing ChIP experiments for OLFM2:

• Crosslinking parameters: Optimize formaldehyde concentration and crosslinking time for capturing OLFM2-associated protein-DNA complexes. Previous studies examining SRF binding to the CArG box of SM22α promoter have successfully implemented ChIP protocols with standard crosslinking parameters .

• Target promoter regions: Focus on promoters of smooth muscle marker genes that contain CArG boxes, particularly the SM22α and SMMHC promoters, which have been validated in previous studies . Design primers that specifically amplify these regions containing the CArG boxes.

• Experimental design: Include appropriate treatments that modify OLFM2 expression or localization. TGF-β treatment significantly enhances SRF binding to CArG boxes in smooth muscle gene promoters, while knockdown of OLFM2 diminishes this enhanced binding .

• Quantification method: qPCR has been used effectively to quantify the enrichment of target promoter regions in ChIP samples . Ensure proper normalization using input samples and negative control regions.

Research has demonstrated that knockdown of OLFM2 significantly diminishes TGF-β-enhanced SRF binding to promoters, while ectopic expression of OLFM2 dose-dependently enhances SRF binding to these promoters even in TGF-β-untreated cells . This approach has revealed that OLFM2 facilitates SRF binding to smooth muscle gene promoters, which is a key event for smooth muscle differentiation .

How can I effectively evaluate OLFM2 antibody specificity for my research?

Ensuring antibody specificity is crucial for obtaining reliable results in OLFM2 research. Based on commercial information and research studies, consider these comprehensive approaches to evaluate OLFM2 antibody specificity:

• Western blot validation: Run samples from multiple species and tissues where OLFM2 is known to be expressed, such as human, mouse, and rat brain tissues . The expected molecular weight range for OLFM2 is 46-51 kDa . Include positive controls (tissues with known OLFM2 expression) and negative controls (OLFM2 knockout tissues or cells with OLFM2 knockdown).

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application to verify that signal disappearance occurs when the antibody's binding sites are blocked by the specific antigen.

• Multiple antibody validation: Compare results using antibodies from different sources or those recognizing different epitopes of OLFM2. Consistent results across different antibodies increase confidence in specificity.

• Genetic models: Utilize samples from OLFM2 knockout or knockdown models as negative controls. Studies have generated OLFM2 deficient mice by replacing the OLFM2 gene with the LacZ gene, which provide excellent negative controls . Similarly, cells treated with OLFM2 shRNA (Ad-shOlfm2) have been used effectively in specificity validation .

• Application-specific validation: For each application (WB, ELISA, IP, etc.), perform specific controls relevant to that technique. For example, in IP experiments, include isotype control antibodies and verify results with reverse IP using known interaction partners .

Commercial OLFM2 antibodies have been validated against human, mouse, and rat samples, with specific reactivity confirmed in brain tissues . The recommended dilution ranges (1:500-1:3000 for WB) provide a starting point, but optimization in your specific experimental system is essential for optimal results .

How do I interpret changes in OLFM2 expression during TGF-β-induced differentiation?

Interpreting changes in OLFM2 expression during TGF-β-induced differentiation requires understanding of both temporal patterns and cellular localization. Research has revealed several key patterns that should be considered when analyzing your experimental data:

• Temporal expression pattern: OLFM2 expression increases markedly soon after TGF-β induction and shows time-dependent upregulation, with studies documenting an 8.4-fold increase after 48 hours of treatment . Importantly, OLFM2 induction occurs earlier than the expression of smooth muscle markers such as α-SMA, SM22α, and SMMHC . This temporal sequence suggests OLFM2 functions as an early mediator in the differentiation process.

• Subcellular localization shifts: Before TGF-β treatment, OLFM2 is distributed in both cytoplasm and nuclei, with approximately 38% located in the nuclei . After TGF-β stimulation, there is a significant redistribution, with nuclear OLFM2 increasing to 81% of the total protein . This nuclear accumulation indicates OLFM2's role in transcriptional regulation during differentiation.

• Correlation with SM markers: A positive correlation should be observed between OLFM2 upregulation and subsequent increases in smooth muscle markers . If this correlation is absent, it may indicate interference in the signaling pathway.

• Functional significance: To properly interpret OLFM2 expression changes, functional studies are essential. For example, knockdown of OLFM2 significantly attenuates TGF-β-induced SM marker expression, while ectopic expression of OLFM2 alone induces increases in α-SMA (3.2-fold), SM22α (2.3-fold), and SMMHC (3.6-fold) expression .

When troubleshooting unexpected results, consider the following: insufficient TGF-β concentration or activity, timing of sample collection that might miss peak expression, cell-type specific responses, or interference from other signaling pathways. Additionally, ensure that your antibody detection method has sufficient sensitivity to detect the range of OLFM2 expression changes.

What are the possible explanations for discrepancies in OLFM2 molecular weight observed in Western blots?

Discrepancies in OLFM2 molecular weight observed in Western blots can arise from multiple biological and technical factors. The calculated molecular weight of OLFM2 is 51 kDa based on its 454 amino acid sequence, but the observed molecular weight typically ranges from 46-51 kDa . When troubleshooting such discrepancies, consider these possible explanations:

• Post-translational modifications:

  • OLFM2 is a secretory glycoprotein , and varying degrees of glycosylation can significantly affect its apparent molecular weight

  • Other modifications such as phosphorylation, ubiquitination, or proteolytic processing may alter migration patterns

• Protein isoforms:

  • Alternative splicing may generate different OLFM2 isoforms with distinct molecular weights

  • Species-specific variations might exist between human, mouse, and rat OLFM2

• Technical factors:

  • Running buffer composition and pH can influence protein migration

  • Gel percentage and type (gradient vs. fixed percentage) affect resolution

  • Sample preparation methods, particularly heating conditions, can impact protein conformation and SDS binding

  • Reducing vs. non-reducing conditions may reveal different patterns if disulfide bonds are present

• Sample-specific considerations:

  • Tissue-specific modifications may result in different apparent weights (e.g., brain-derived OLFM2 vs. smooth muscle-derived OLFM2)

  • Nuclear vs. cytoplasmic fractions might reveal differently modified forms of OLFM2

To address these discrepancies, compare your results with published literature reporting OLFM2 Western blot findings in similar experimental contexts. For instance, studies have successfully detected OLFM2 in human brain tissue, fetal human brain tissue, mouse brain tissue, and rat brain tissue within the expected molecular weight range . Additionally, consider running controls from different tissues/species alongside your samples and potentially employing epitope mapping to determine if your antibody recognizes specific domains or modifications of OLFM2.

How can I analyze OLFM2's role in protein complexes using biochemical fractionation approaches?

Analyzing OLFM2's role in protein complexes requires sophisticated biochemical fractionation approaches that preserve native interactions while allowing effective separation of different cellular compartments. Based on published research, consider this methodological framework:

• Synaptosomal fractionation for neurological studies:
  OLFM2 co-segregates with AMPA receptor complexes in synaptosomal membrane fractions from brain cortex and retina . This approach has successfully demonstrated that OLFM2 interacts with the GluR2 subunit of AMPAR complexes and associates with other synaptic proteins . The technique involves:

  • Sequential centrifugation steps to isolate synaptosomal fractions

  • Careful membrane solubilization to maintain protein complexes

  • Density gradient separation for further purification

• Nuclear-cytoplasmic fractionation for TGF-β signaling studies:
  Given that TGF-β induces nuclear accumulation of OLFM2 (from 38% to 81% nuclear localization) , nuclear-cytoplasmic fractionation is essential for studying OLFM2's role in transcriptional regulation. This approach should:

  • Employ gentle lysis conditions to preserve nuclear integrity

  • Include careful washing steps to prevent cross-contamination

  • Verify fractionation quality with compartment-specific markers

• Complex analysis through co-immunoprecipitation:
  After fractionation, co-immunoprecipitation from specific fractions can reveal interaction partners. Key findings from previous studies include:

  • Immunoprecipitation from synaptosomal membrane fractions of OLFM2 null mouse brain cortex using GluR2 antibody showed reduced levels of AMPAR complex components including Olfm1, PSD95, and CNIH2

  • OLFM2 physically interacts with SRF, and this interaction is enhanced by TGF-β treatment

  • OLFM2 regulates SRF's interaction with HERP1, a transcriptional repressor

• Quantitative analysis of complex components:
  Western blotting with densitometric analysis can quantify changes in complex composition under different conditions:

  • Compare wild-type vs. OLFM2 knockout samples

  • Analyze TGF-β treated vs. untreated samples

  • Assess OLFM2 overexpression effects

Research suggests that heterodimers of Olfm1 and Olfm2 interact with AMPAR more efficiently than Olfm2 homodimers , highlighting the importance of analyzing protein complex stoichiometry. Additionally, OLFM2 has been shown to mediate TGF-β-induced dissociation of HERP1 from SRF, a key mechanism in smooth muscle differentiation .

What phenotypes are associated with OLFM2 deficiency in animal models?

OLFM2 deficiency in animal models reveals important insights into its physiological functions across multiple systems. Studies using OLFM2 knockout mice have identified several distinctive phenotypes:

For researchers studying OLFM2 using animal models, careful behavioral testing protocols should be implemented to detect the subtle but significant phenotypes associated with OLFM2 deficiency. Additionally, molecular analyses focusing on AMPA receptor complex composition and synaptic function would be valuable for understanding the mechanistic basis of these phenotypic changes.

How can I design experiments to study OLFM2's role in smooth muscle differentiation?

Designing experiments to study OLFM2's role in smooth muscle differentiation requires a multifaceted approach that captures both molecular mechanisms and functional outcomes. Based on published research, consider this comprehensive experimental framework:

• Cell model selection:

  • Human embryonic stem cell-derived mesenchymal cells (hES-MCs) have been successfully used to study TGF-β-induced smooth muscle differentiation and OLFM2's role in this process

  • Primary smooth muscle cells or progenitor cells can provide complementary insights

  • Consider including smooth muscle cells from different vascular beds to assess tissue-specific effects

• Key experimental manipulations:

  • Gain-of-function studies: Implement ectopic expression of OLFM2 using adenoviral or lentiviral vectors to assess whether OLFM2 alone can induce smooth muscle marker expression

  • Loss-of-function studies: Use shRNA-mediated knockdown (e.g., Ad-shOlfm2) to attenuate OLFM2 expression and evaluate its necessity for TGF-β-induced differentiation

  • Treatment conditions: Apply TGF-β treatment (typically 5 ng/ml) for varying time periods (12h, 24h, 48h) to track the temporal relationship between OLFM2 induction and smooth muscle marker expression

• Essential readouts:

  • Gene expression analysis: Measure mRNA levels of smooth muscle markers (α-SMA, SM22α, SMMHC) using qRT-PCR

  • Protein expression: Assess protein levels of smooth muscle markers using Western blotting

  • Subcellular localization: Track OLFM2 nuclear accumulation using immunofluorescence and subcellular fractionation approaches

  • Protein-protein interactions: Examine OLFM2-SRF interaction using co-immunoprecipitation

  • DNA-protein interactions: Assess SRF binding to CArG boxes in smooth muscle gene promoters using ChIP assays

• Functional assessments:

  • Contractility assays to measure functional differentiation

  • Cell morphology assessment to track phenotypic changes

  • Migration and proliferation assays to evaluate changes in cell behavior

Research has demonstrated that OLFM2 regulates smooth muscle differentiation through several mechanisms:

• Physical interaction with SRF to counteract the association between SRF and HERP1 (a transcriptional repressor)

• Enhancement of SRF binding to CArG boxes in smooth muscle gene promoters

• Inhibition of HERP1 expression

These molecular mechanisms provide multiple points for experimental intervention and assessment when designing a comprehensive study of OLFM2's role in smooth muscle differentiation.