osr2 Antibody

What is OSR2 and why is it significant in research?

OSR2 (Odd-Skipped Related 2) is a transcription factor belonging to the Odd C2H2-type zinc-finger protein family. In humans, the canonical protein consists of 312 amino acid residues with a molecular mass of approximately 35.5 kDa and localizes to the nucleus . OSR2 plays a crucial role in cellular differentiation processes, making it a significant target for developmental biology and disease research . Up to three different isoforms have been reported for this protein, suggesting diverse functionality depending on cellular context . The gene is highly conserved, with orthologs identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, indicating its evolutionary importance in vertebrate development .

How do I select the appropriate OSR2 antibody for my experimental needs?

Selection of an appropriate OSR2 antibody depends on multiple factors including:

• Target region specificity: Determine whether you need an antibody targeting the N-terminal, internal region, or specific amino acid sequences (e.g., AA 101-276) . This choice depends on protein accessibility in your experimental conditions and whether you need to distinguish between isoforms.

• Species reactivity: Verify the antibody's reactivity with your species of interest. Available antibodies show varying cross-reactivity profiles:

  Antibody Type	Species Reactivity	Cross-Reactivity Percentage
  N-Terminal (ABIN2777344)	Human, Mouse, Rat, Cow, Dog, Guinea Pig, Horse, Rabbit	Human: 100%, Mouse: 93%, Rat: 93%, Cow: 93%, Dog: 93%, Guinea Pig: 93%, Horse: 93%, Rabbit: 93%
  Internal Region (ABIN6258294)	Human, Mouse, Rat	Not specified
  Middle Region (ARP90990_P050)	Mouse	Not specified

• Application compatibility: Different antibodies are optimized for specific techniques such as Western Blot (WB), ELISA, Immunohistochemistry (IHC), Immunocytochemistry (ICC), or Immunofluorescence (IF) . Choose one validated for your intended application.

• Clonality consideration: Most available OSR2 antibodies are polyclonal (from rabbit) , which offers high sensitivity but potentially lower specificity than monoclonal alternatives.

What are the common applications for OSR2 antibodies in research?

OSR2 antibodies are employed in multiple research applications with varying degrees of technical complexity:

• Western Blotting (WB): Most commonly used application for detecting OSR2 protein expression levels and molecular weight confirmation .

• Enzyme-Linked Immunosorbent Assay (ELISA): Used for quantitative assessment of OSR2 levels in samples .

• Immunohistochemistry (IHC): For visualizing OSR2 distribution in tissue sections, particularly useful in developmental studies and pathological examinations .

• Immunocytochemistry (ICC): Allows subcellular localization studies of OSR2 in cultured cells .

• Immunofluorescence (IF): Provides high-resolution imaging of OSR2 distribution, often used in co-localization studies with other proteins .

• Immunoprecipitation (IP): Used for isolating OSR2 protein complexes to study protein-protein interactions .

How can I optimize Western blot protocols for detecting low-abundance OSR2 isoforms?

Optimizing Western blot protocols for low-abundance OSR2 isoforms requires multiple technical considerations:

• Sample preparation enhancement:

  • Employ nuclear extraction protocols given OSR2's nuclear localization

  • Utilize phosphatase and protease inhibitors freshly added to lysis buffers

  • Increase protein loading (50-100 μg) for low-abundance isoforms

• Detection sensitivity improvement:

  • Select antibodies targeting unique epitopes in specific isoforms, such as the N-terminal antibody (ABIN2777344) which recognizes the sequence "YSFLQAVNTF PATVDHLQGL YGLSAVQTMH MNHWTLGYPN VHEITRSTIT"

  • Employ enhanced chemiluminescence (ECL) substrates with extended signal duration

  • Consider signal amplification systems (tyramide or biotin-based)

• Transfer optimization:

  • For the 35.5 kDa OSR2 protein, use PVDF membranes with 0.2 μm pore size

  • Implement wet transfer at lower voltage (30V) overnight at 4°C

  • Use transfer buffers containing 10-20% methanol for proteins of this size range

• Blocking and antibody incubation:

  • Test alternative blocking agents (5% BSA often works better than milk for phosphorylated proteins)

  • Increase primary antibody incubation time (overnight at 4°C) with optimized dilution

  • Consider validated antibodies like those affinity-purified via peptide chromatography or synthetic peptide immunogens

What are the critical considerations when using OSR2 antibodies for immunohistochemistry in different tissue types?

When performing immunohistochemistry with OSR2 antibodies across tissue types, researchers should address several critical factors:

• Tissue-specific fixation optimization:

  • For embryonic tissues where OSR2 plays developmental roles: use 4% paraformaldehyde for 24 hours at 4°C

  • For adult tissues: test both paraformaldehyde and formalin fixation in parallel

  • Consider antigen retrieval requirements based on epitope accessibility in your tissue type

• Antibody selection based on tissue specificity:

  • For cross-species studies, select antibodies with demonstrated reactivity across species of interest

  • The internal region antibody (ABIN6258294) shows reactivity with human, mouse, and rat tissues

  • For mouse-specific studies, consider the middle region antibody (ARP90990_P050)

• Background reduction strategies:

  • Implement tissue-specific blocking protocols (10% serum from the secondary antibody species)

  • Include avidin/biotin blocking steps when using biotinylated detection systems

  • Consider autofluorescence quenching methods for certain tissues (liver, brain)

• Controls implementation:

  • Always include negative controls (primary antibody omission, isotype controls)

  • When possible, include tissues from OSR2 knockout models as gold-standard negative controls

  • Use tissues with known high OSR2 expression as positive controls

• Signal development optimization:

  • For chromogenic detection: optimize development times based on tissue type

  • For fluorescent detection: consider signal amplification for tissues with low OSR2 expression

  • Consider multiplex staining to contextualize OSR2 expression with lineage markers

How do post-translational modifications affect OSR2 antibody binding efficiency?

Post-translational modifications (PTMs) can significantly influence OSR2 antibody recognition and binding efficiency through several mechanisms:

• Impact on epitope accessibility:

  • Phosphorylation events may alter protein conformation, potentially masking or exposing antibody binding sites

  • C2H2 zinc-finger domains (present in OSR2) are known to undergo SUMOylation which can interfere with antibody binding

  • Consider using phosphatase treatment in parallel samples to assess phosphorylation impact

• Modification-specific detection strategies:

  • For studies focused on OSR2 PTMs, consider using:

    • Antibodies targeting specific modification sites (if available)

    • Combination of general OSR2 antibodies with PTM-specific antibodies in co-IP experiments

    • Pretreatment of samples with modification-removing enzymes to compare detection levels

• Antibody selection considerations:

  • Antibodies targeting different regions might have varying sensitivity to PTMs

  • The internal region antibody (ABIN6258294) targets a larger region (likely spanning multiple potential modification sites)

  • The N-terminal antibody (ABIN2777344) targets a more defined sequence which might have fewer modification sites

• Experimental design adaptation:

  • Include appropriate controls for PTM studies (phosphatase-treated, deglycosylated samples)

  • Consider using multiple antibodies targeting different epitopes to confirm results

  • Implement mass spectrometry validation of PTM status when possible

What are the optimal storage conditions for maintaining OSR2 antibody activity long-term?

Maintaining OSR2 antibody activity requires careful attention to storage conditions:

• Temperature considerations:

  • Long-term storage: -20°C to -80°C in non-frost-free freezers

  • Working aliquots: 4°C for up to 2 weeks

  • Avoid repeated freeze-thaw cycles (create 10-20 μL single-use aliquots)

• Buffer composition impact:

  • Most commercial OSR2 antibodies are supplied in buffered aqueous glycerol solutions

  • Glycerol percentage (typically 30-50%) prevents freezing at -20°C

  • Presence of preservatives like sodium azide (0.02-0.05%) inhibits microbial growth

  • Some antibodies undergo buffer exchange during affinity purification processes

• Physical handling precautions:

  • Store upright to prevent cap contamination

  • Centrifuge briefly before opening to collect solution at bottom

  • Use sterile techniques when handling to prevent contamination

  • Consider oxygen-free environments for highly sensitive antibodies

• Stability monitoring protocols:

  • Implement regular activity testing on control samples

  • Document lot-to-lot variation with standardized positive controls

  • Consider adding carrier proteins (BSA 1-5 mg/mL) to dilute antibody solutions

What validation steps should be performed when using OSR2 antibodies for the first time?

When introducing a new OSR2 antibody into your research, comprehensive validation is essential:

How can I determine the optimal antibody concentration for different experimental applications?

Determining optimal antibody concentration requires systematic titration across applications:

• Western blot titration approach:

  • Start with manufacturer's recommended dilution range

  • Test 3-5 dilutions in 2-fold or 5-fold increments (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Evaluate signal-to-background ratio, not just signal intensity

  • Recommended starting dilutions for many OSR2 antibodies are 1:500-1:1000

• Immunohistochemistry optimization:

  • For paraffin sections: Begin with higher concentrations (1:50-1:200)

  • For frozen sections: Start with more dilute solutions (1:200-1:500)

  • Include antigen retrieval method optimization in parallel

  • Assess non-specific binding in negative control tissues

• ELISA concentration determination:

  • Perform checkerboard titration (antibody vs. standard protein)

  • Calculate signal-to-noise ratio at each concentration

  • Determine lower limit of detection and quantification

  • Select concentration that provides linear response in your working range

• Immunofluorescence considerations:

  • Start with manufacturer's recommendation for IF applications

  • For polyclonal OSR2 antibodies used in IF , typically begin at 1:100-1:500

  • Adjust based on microscopy system sensitivity and autofluorescence levels

  • Balance signal intensity with background and non-specific binding

How can I address non-specific binding when using OSR2 antibodies in immunofluorescence studies?

Non-specific binding in OSR2 immunofluorescence can be systematically addressed:

• Blocking optimization strategies:

  • Extend blocking time (1-2 hours at room temperature)

  • Test alternative blocking agents (5% BSA, 10% serum, commercial blockers)

  • Add 0.1-0.3% Triton X-100 for better antibody penetration

  • Include protein blockers specific to your secondary antibody species

• Antibody incubation refinement:

  • Dilute antibodies in blocking buffer containing 1-3% blocking protein

  • Increase wash steps (5-6 washes of 5-10 minutes each)

  • Consider overnight incubation at 4°C instead of room temperature

  • For OSR2 antibodies from affinity purification processes , additional dilution may improve specificity

• Technical optimization approaches:

  • Pre-adsorb antibody with tissue powder from negative control samples

  • Implement more stringent washing conditions (higher salt, mild detergents)

  • Consider using highly cross-adsorbed secondary antibodies

  • Reduce secondary antibody concentration if background remains high

• Controls and validation:

  • Always include secondary-only controls to assess non-specific binding

  • Implement peptide competition controls with the immunizing peptide

  • Compare staining pattern with multiple OSR2 antibodies targeting different epitopes

  • Correlate with other detection methods (WB, IHC) to confirm specificity

What are the most common causes of inconsistent OSR2 antibody performance in Western blots?

Inconsistent OSR2 antibody performance in Western blots can stem from multiple factors:

• Sample preparation variables:

  • Incomplete protein denaturation (OSR2 is a nuclear protein with potential protein-DNA interactions)

  • Inadequate lysis (nuclear proteins require specialized extraction)

  • Protein degradation (ensure fresh protease inhibitors)

  • Variable sample loading (verify with housekeeping controls)

• Technical execution issues:

  • Inconsistent transfer efficiency (verify with reversible staining)

  • Variable blocking efficiency (standardize blocking time/temperature)

  • Antibody degradation (aliquot and store properly)

  • Inconsistent ECL reagent performance (prepare fresh)

• Antibody-specific factors:

  • Lot-to-lot variation (maintain records of performance by lot)

  • Freeze-thaw degradation (create single-use aliquots)

  • Non-optimal dilution (re-optimize with each new lot)

  • Target epitope accessibility issues (try multiple antibodies targeting different regions)

• Resolution strategies:

  • Implement standardized protocols with detailed documentation

  • Include positive control samples with known OSR2 expression

  • Consider including recombinant OSR2 protein as reference standard

  • When switching antibody lots, run side-by-side comparison with previous lot

How should I interpret conflicting data between different OSR2 antibodies in the same experiment?

When faced with conflicting results from different OSR2 antibodies, follow this interpretation framework:

• Antibody characteristic assessment:

  • Compare targeting epitopes (N-terminal vs. internal region antibodies may detect different isoforms)

  • Review validation data from manufacturers for each antibody

  • Consider clonality differences (all reported OSR2 antibodies are polyclonal but may target different epitopes)

  • Assess purification methods (peptide affinity chromatography vs. affinity purified )

• Experimental condition analysis:

  • Evaluate whether conditions favor one epitope's accessibility over others

  • Consider whether sample preparation might differentially affect epitope integrity

  • Assess whether detection methods have varied sensitivity thresholds

• Biological interpretation strategies:

  • Consider the possibility of detecting different OSR2 isoforms (up to 3 reported)

  • Evaluate whether post-translational modifications might affect epitope recognition

  • Assess potential cross-reactivity with related proteins

• Resolution approaches:

  • Implement orthogonal validation methods (mass spectrometry, RNA expression)

  • Utilize genetic approaches (siRNA, CRISPR) to confirm specificity

  • Consider the collective weight of evidence rather than relying on a single antibody

  • Transparently report conflicting results in publications with potential interpretations

How can OSR2 antibodies be employed in developmental biology research?

OSR2 antibodies offer valuable tools for developmental biology studies:

• Spatiotemporal expression profiling:

  • Map OSR2 expression patterns during embryonic development using IHC/IF

  • Track dynamic changes across developmental stages

  • Correlate with morphological events and other developmental markers

  • The nuclear localization of OSR2 provides clear subcellular resolution for developmental studies

• Lineage tracing applications:

  • Combine OSR2 immunostaining with lineage-specific markers

  • Track cell fate decisions in OSR2-expressing populations

  • Implement dual/triple immunofluorescence with other transcription factors

  • Correlate with functional outcomes in developmental processes

• Mechanistic developmental studies:

  • Use OSR2 antibodies in ChIP assays to identify target genes

  • Implement Co-IP to identify protein interaction partners during development

  • Study regulation of OSR2 by upstream developmental signals

  • Investigate post-translational modifications during developmental transitions

• Cross-species developmental comparisons:

  • Leverage the cross-reactivity of certain OSR2 antibodies with multiple species

  • Compare expression patterns across evolutionary distance

  • Study conservation of developmental programs involving OSR2

  • Correlate with evolutionary adaptations in different species

What methodological advances are emerging for studying OSR2 protein interactions?

Emerging methodologies for studying OSR2 protein interactions include:

• Advanced proximity labeling approaches:

  • BioID or TurboID fusion with OSR2 to identify proximal proteins in living cells

  • APEX2-based proximity labeling for temporal resolution of interaction networks

  • Split-BioID systems to study context-dependent interactomes

  • These methods can be validated using traditional Co-IP with OSR2 antibodies

• Live-cell interaction monitoring:

  • FRET/BRET with fluorescently tagged OSR2 and candidate interactors

  • BiFC (Bimolecular Fluorescence Complementation) for direct visualization

  • Single-molecule tracking to study dynamics of interactions

  • Correlate with fixed-cell immunofluorescence using OSR2 antibodies

• High-throughput interaction screening:

  • IP-mass spectrometry workflows using OSR2 antibodies for immunoprecipitation

  • Protein microarray screening with recombinant OSR2

  • Yeast two-hybrid or mammalian two-hybrid screening

  • Validation of hits using reciprocal Co-IP with OSR2 antibodies

• Structural studies of interactions:

  • Cryo-EM of OSR2-containing complexes purified via immunoprecipitation

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cross-linking mass spectrometry to identify spatial proximity

  • Correlation with antibody epitope mapping data to understand functional domains

How can OSR2 antibodies contribute to understanding pathological conditions?

OSR2 antibodies provide valuable tools for investigating pathological conditions:

• Cancer research applications:

  • Evaluate OSR2 expression changes in tumor tissues via IHC

  • Correlate expression with clinical outcomes and tumor characteristics

  • Study mechanisms of dysregulation using cell line models

  • Investigate potential as a diagnostic or prognostic biomarker

• Developmental disorder studies:

  • Analyze OSR2 expression in tissues from developmental disorder models

  • Correlate with phenotypic manifestations in animal models

  • Investigate human patient samples when available

  • The zinc-finger protein family (including OSR2) has established roles in development

• Tissue regeneration and wound healing research:

  • Monitor OSR2 expression during tissue repair processes

  • Investigate role in cell differentiation during regeneration

  • Study potential regulatory functions in stem cell populations

  • Correlate with functional recovery outcomes

• Mechanistic studies in disease models:

  • Implement ChIP-seq using OSR2 antibodies to identify altered gene targeting

  • Study protein-protein interaction changes in disease states via Co-IP

  • Investigate post-translational modification alterations in pathological conditions

  • Correlate with functional consequences and potential therapeutic implications