OFP15 Antibody

What is OFP15 Antibody and what is its specific target?

OFP15 Antibody is a rabbit polyclonal antibody that specifically targets the OFP15 protein (UniProt Number: Q9SJ45) from Arabidopsis thaliana (Mouse-ear cress). The antibody is developed using a recombinant Arabidopsis thaliana OFP15 protein as the immunogen . As a research reagent, it's designed for the specific detection of OFP15 protein in experimental settings such as immunoassays and protein analysis techniques.

The antibody preparation typically contains three components:

• 200μg antigens (positive control)

• 1ml pre-immune serum (negative control)

• Rabbit polyclonal antibodies purified by Antigen Affinity

What applications is OFP15 Antibody validated for?

OFP15 Antibody has been validated for the following applications:

The antibody is particularly useful in plant biology research focusing on Arabidopsis thaliana, which serves as a model organism for understanding plant molecular biology and genetics .

How should researchers validate OFP15 Antibody for their specific experiments?

When validating OFP15 Antibody for your research, follow these methodological steps:

• Positive controls: Use the provided antigen (200μg) as a positive control to confirm antibody binding specificity .

• Negative controls:

  • Utilize the included pre-immune serum as a negative control

  • Test with samples known to lack OFP15 expression

  • Include secondary antibody-only controls to detect non-specific binding

• Specificity testing: Verify antibody specificity using knockout or knockdown systems if available. This is crucial as research has shown that many commercial antibodies may bind to proteins other than their intended targets, potentially leading to misleading results .

• Western blot validation: Run a dilution series to determine optimal antibody concentration. Look for a single band at the expected molecular weight of OFP15 protein.

• Cross-reactivity assessment: Test the antibody against related proteins to ensure it does not cross-react with other OFP family members or structurally similar proteins .

What are the specificity and sensitivity metrics for OFP15 Antibody?

While specific values for OFP15 Antibody are not provided in the available data, understanding antibody specificity and sensitivity is critical for experimental design. For reference, high-quality antibodies typically demonstrate:

• Specificity: The ability to distinguish the target antigen from other proteins. For research-grade antibodies, specificity above 99% is considered excellent, similar to diagnostic antibody tests which demonstrate specificity of 99.56-99.63% .

• Sensitivity: The ability to detect low concentrations of the target protein. Quality research antibodies should detect their targets when present at physiologically relevant concentrations, comparable to diagnostic antibodies which have sensitivities of 95-100% when tested at appropriate timepoints .

Researchers should perform their own validation to establish these metrics for their specific experimental conditions. This is particularly important given recent findings of significant issues with antibody specificity in research applications .

How can researchers optimize Western blot protocols for OFP15 Antibody?

For optimal Western blot results with OFP15 Antibody, consider these methodological adjustments:

• Sample preparation:

  • Use appropriate extraction buffers optimized for plant tissues

  • Include protease inhibitors to prevent degradation

  • Ensure complete denaturation of samples if required for epitope accessibility

• Blocking optimization:

  • Test both BSA and non-fat dry milk as blocking agents

  • Consider 5% blocking solution in TBS-T (Tris-buffered saline with 0.1% Tween-20)

  • Block for 1-2 hours at room temperature or overnight at 4°C

• Antibody dilution optimization:

  • Start with a dilution series (1:500, 1:1000, 1:2000, 1:5000)

  • Incubate primary antibody overnight at 4°C for optimal binding

• Signal development:

  • Choose detection methods based on expected expression levels

  • Consider enhanced chemiluminescence for standard detection

  • Use fluorescent secondary antibodies for multiplexing or quantitative analysis

• Controls:

  • Always include the provided positive control antigen

  • Use the pre-immune serum as a negative control

What factors affect epitope accessibility when using OFP15 Antibody in different applications?

Several factors can affect epitope accessibility and recognition by OFP15 Antibody:

• Protein conformation: The three-dimensional structure of the target protein can mask epitopes. Consider using both native and denaturing conditions when optimizing protocols.

• Fixation methods: Different fixation methods can preserve or destroy epitopes:

  • Paraformaldehyde may preserve structure but mask epitopes

  • Methanol fixation may expose some epitopes while destroying others

  • Heat-induced epitope retrieval may be necessary for some applications

• Post-translational modifications: PTMs such as phosphorylation, glycosylation, or ubiquitination may alter epitope recognition. Consider the physiological state of your samples.

• Sample preparation: Harsh detergents or extraction methods may denature the protein or damage epitopes.

• pH and buffer conditions: Buffer composition can affect antibody-antigen interactions. Test different pH conditions and buffer compositions to optimize binding.

How should researchers address potential cross-reactivity with other OFP family proteins?

Plant OVATE Family Proteins (OFPs) share structural similarities that may lead to cross-reactivity. To address this:

• Preabsorption controls: Preincubate the antibody with purified related proteins to absorb cross-reactive antibodies.

• Knockout/knockdown validation: Test the antibody in tissues/cells with confirmed knockout or knockdown of OFP15.

• Western blot analysis: Compare band patterns between wild-type and OFP15-deficient samples.

• Bioinformatic analysis: Identify regions of sequence similarity between OFP15 and other OFP family members to predict potential cross-reactivity.

• Competitive binding assays: Perform competition experiments with peptides representing unique and conserved regions of OFP15.

What are common sources of false positives/negatives when using OFP15 Antibody?

False results can arise from several sources:

False Positives:

• Cross-reactivity with related proteins

• Non-specific binding to high-abundance proteins

• Insufficient blocking

• Excessive antibody concentration

• Secondary antibody cross-reactivity

False Negatives:

• Epitope masking due to protein conformation

• Protein degradation during sample preparation

• Insufficient protein transfer in Western blots

• Improper storage leading to antibody degradation

• Interference from sample components

How can researchers quantitatively assess OFP15 Antibody binding characteristics?

To quantitatively characterize OFP15 Antibody:

• Surface Plasmon Resonance (SPR): Determine binding kinetics including:

  • Association rate constant (kon)

  • Dissociation rate constant (koff)

  • Equilibrium dissociation constant (KD)

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Perform titration curves to determine EC50 values

  • Compare binding to positive and negative controls

• Immunoprecipitation efficiency:

  • Quantify the percentage of target protein pulled down

  • Analyze specific vs. non-specific binding

• Western blot quantification:

  • Generate standard curves using purified recombinant protein

  • Use densitometry to quantify signals

Recent high-throughput approaches for characterizing antibody variants could also be applied to better understand OFP15 Antibody binding properties .

What are the recommended storage conditions to maintain OFP15 Antibody stability?

To ensure optimal stability and performance of OFP15 Antibody:

• Storage temperature: Store at -20°C or -80°C for long-term stability .

• Aliquoting: Divide into small, single-use aliquots to avoid repeated freeze-thaw cycles.

• Additives: Consider adding stabilizers such as:

  • Glycerol (50%) for freeze protection

  • BSA (1 mg/ml) to prevent adsorption to tube walls

  • Preservatives like sodium azide (0.02%) to prevent microbial growth

• Working dilutions: Store working dilutions at 4°C for up to one week; for longer periods, store at -20°C.

• Stability monitoring: Periodically test antibody performance against a reference standard to monitor potential degradation.

How can OFP15 Antibody be used in immunoprecipitation studies of plant protein complexes?

For immunoprecipitation (IP) applications:

• Sample preparation:

  • Use gentle lysis buffers to maintain protein-protein interactions

  • Include protease and phosphatase inhibitors

  • Perform pre-clearing with pre-immune serum to reduce non-specific binding

• Antibody coupling:

  • Consider covalently coupling OFP15 Antibody to protein A/G beads

  • Use crosslinkers that minimize antibody leaching during elution

• Optimization strategies:

  • Test different antibody-to-lysate ratios

  • Optimize binding conditions (temperature, duration, buffer composition)

  • Include appropriate controls (pre-immune serum, IgG control)

• Validation:

  • Confirm specificity by Western blot analysis of immunoprecipitated material

  • Verify enrichment of target protein compared to input sample

• Downstream applications:

  • Mass spectrometry analysis of co-immunoprecipitated proteins

  • Functional assays of immunoprecipitated complexes

What methodological considerations are important when using OFP15 Antibody in chromatin immunoprecipitation studies?

For chromatin immunoprecipitation (ChIP) applications:

• Crosslinking optimization:

  • Test different crosslinking times and concentrations

  • Consider dual crosslinking with both formaldehyde and protein-specific crosslinkers

• Chromatin fragmentation:

  • Optimize sonication conditions for plant tissues

  • Aim for fragments between 200-500 bp for optimal resolution

• Antibody specificity:

  • Verify OFP15 localization to chromatin or nuclear compartments

  • Include appropriate controls (IgG, pre-immune serum)

• Washing stringency:

  • Optimize salt concentrations in wash buffers

  • Balance between reducing background and maintaining specific interactions

• Data analysis:

  • Use appropriate normalization strategies

  • Compare enrichment to input and negative control regions

Note that these applications require additional validation beyond the standard ELISA and Western blot applications listed in the product specifications .

How does the polyclonal nature of OFP15 Antibody affect experimental design considerations?

The polyclonal nature of OFP15 Antibody has important implications:

Advantages to consider:

• Recognition of multiple epitopes, increasing detection sensitivity

• More robust to minor changes in protein conformation

• Greater tolerance to sample preparation variations

• Often works across multiple applications (WB, ELISA, IP)

Limitations to address:

• Batch-to-batch variation requires validation between lots

• Greater potential for cross-reactivity with related proteins

• Higher background in some applications

• Less predictable epitope specificity

Methodological recommendations:

• Compare critical experiments across antibody lots

• Include comprehensive controls for cross-reactivity

• Consider epitope mapping to identify the regions recognized

• For quantitative applications, establish standard curves with each new lot

How does OFP15 Antibody compare with alternative methods for detecting OFP15 expression?

When selecting detection methods, consider these comparisons:

This comparison highlights the importance of choosing the appropriate method based on your specific research question and experimental requirements.

What are best practices for validating new lots of OFP15 Antibody?

Lot-to-lot validation is critical for polyclonal antibodies. Follow these best practices:

• Record keeping:

  • Document lot numbers

  • Retain reference samples from previous lots

• Side-by-side comparison:

  • Test new and old lots in parallel

  • Use identical samples and protocols

• Standard curve analysis:

  • Generate dilution series with purified antigen

  • Compare EC50 or limit of detection values

• Specificity assessment:

  • Test against positive and negative control samples

  • Check for unexpected bands or staining patterns

• Performance metrics:

  • Compare signal-to-noise ratios

  • Evaluate background levels

  • Assess specificity in relevant experimental contexts

This approach aligns with recent efforts to systematically evaluate antibodies used in research, addressing the concerning finding that many antibodies may not specifically bind their intended targets .

How might advanced characterization techniques improve understanding of OFP15 Antibody binding properties?

Emerging technologies offer opportunities for enhanced antibody characterization:

• Structural analysis:

  • Epitope mapping using hydrogen-deuterium exchange mass spectrometry

  • X-ray crystallography of antibody-antigen complexes

  • Cryo-electron microscopy for structural determination

• High-throughput binding analysis:

  • Sensor-integrated Proteome On Chip (SPOC®) platform for measuring kinetic binding rates

  • Microfluidic antibody-binding assays

  • Single-molecule imaging of binding events

• Computational prediction:

  • Molecular surface descriptors to predict binding characteristics

  • Machine learning approaches for epitope prediction

  • Molecular dynamics simulations of antibody-antigen interactions

• Next-generation sequencing:

  • Repertoire analysis of polyclonal antibody populations

  • Identification of dominant epitope-specific clones

These approaches could provide deeper insights into OFP15 Antibody binding properties and guide optimization of experimental protocols.

What emerging applications might benefit from OFP15 Antibody beyond current research uses?

Potential future applications include:

• Single-cell proteomics:

  • Characterizing OFP15 expression in rare cell populations

  • Spatial transcriptomics combined with antibody detection

• CRISPR screening validation:

  • Confirming protein-level changes in gene-edited plants

  • Assessing phenotypic consequences of OFP15 modifications

• Synthetic biology applications:

  • Engineering plant signaling pathways involving OFP proteins

  • Developing biosensors for monitoring plant stress responses

• Directed evolution approaches:

  • Using active learning to improve antibody-antigen binding prediction

  • Developing enhanced OFP15 antibodies with greater specificity

• Translational applications:

  • Agricultural biotechnology applications

  • Plant stress response monitoring in field conditions

These emerging applications highlight the continuing importance of well-characterized, specific antibodies in advancing biological research.