ARF21 Antibody

What is ARF21 and what experimental systems is the antibody validated for?

ARF21 refers to a protein in Arabidopsis thaliana (Mouse-ear cress), a model plant organism in molecular biology research. The antibody against this protein has been validated for ELISA applications according to manufacturer information . ARF21 is identified in biological databases through specific identifiers including KEGG: ath:AT1G34410, STRING: 3702.AT1G34410.1, and UniGene: At.51930 . When designing experiments with this antibody, researchers should consider its validated applications and reactivity with Arabidopsis thaliana samples to ensure proper experimental setup and controls.

What buffer and storage conditions are optimal for maintaining ARF21 antibody activity?

Based on similar research antibodies, ARF21 antibody solutions typically contain preservatives like 0.03% Proclin 300 and are formulated in a stabilizing buffer containing approximately 50% glycerol and 0.01M PBS at pH 7.4. For long-term storage, the antibody should be kept at -20°C or below, with aliquoting recommended to avoid repeated freeze-thaw cycles that can compromise activity. For continuous use over shorter periods (up to one week), undiluted antibody can be stored at 2-8°C . When retrieving from storage, it's advisable to gently mix the solution before use to ensure homogeneity.

How can researchers validate ARF21 antibody specificity in plant tissue samples?

Validation of ARF21 antibody specificity requires a multi-faceted approach:

Rigorous validation is critical as non-specific binding can lead to misinterpretation of experimental results, particularly in complex plant tissue samples.

What are optimal sample preparation protocols for using ARF21 antibody in plant tissue Western blots?

Successful Western blot detection of ARF21 in plant tissues requires careful sample preparation:

• Harvest fresh plant tissue and immediately flash-freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining freezing conditions

• Extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • Protease inhibitor cocktail

  • Optional: 2% polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds

• Clear lysate by centrifugation (14,000 × g, 15 min, 4°C)

• Quantify protein concentration

• Add reducing agent to sample buffer and heat at 95°C for 5 minutes

• Load 20-50 μg total protein per lane

• Following transfer, block membrane with 5% non-fat milk in TBST

• Incubate with ARF21 antibody at optimized dilution (typically starting at 1:1000)

• Develop using chemiluminescence or fluorescent detection systems

This protocol minimizes protein degradation and maximizes specific signal detection while reducing background interference common in plant samples.

How can researchers optimize ELISA conditions for quantitative detection of ARF21?

ELISA has been validated for ARF21 antibody applications . To optimize quantitative detection:

• Coating optimization:

  • Test multiple coating buffers (carbonate-bicarbonate pH 9.6 vs. PBS pH 7.4)

  • Optimize coating concentration (0.5-5 μg/ml) and time (overnight at 4°C vs. 2 hours at 37°C)

• Blocking optimization:

  • Compare BSA (1-5%) vs. non-fat dry milk (1-5%)

  • Evaluate blocking time (1-3 hours) and temperature (RT vs. 37°C)

• Antibody dilution matrix:

  • Prepare a dilution series of primary antibody (1:500 to 1:5000)

  • Test with a range of secondary antibody dilutions (1:1000 to 1:10000)

  • Include calibration standards if purified ARF21 protein is available

• Detection system optimization:

  • Compare colorimetric (TMB substrate) vs. chemiluminescent detection

  • Optimize development time (5-30 minutes) for optimal signal-to-noise ratio

• Validation controls:

  • Include wild-type and arf21 mutant extracts as positive and negative controls

  • Prepare standard curves using recombinant ARF21 if available

The optimized protocol should yield a linear standard curve with R² > 0.98 and coefficient of variation < 10% between technical replicates.

What strategies can minimize cross-reactivity when using ARF21 antibody in immunohistochemistry?

Cross-reactivity is a common challenge in plant immunohistochemistry. To minimize this issue:

• Sample preparation refinements:

  • Fix tissues in 4% paraformaldehyde for precisely controlled duration (2-4 hours)

  • Perform careful dehydration and embedding to preserve protein antigenic sites

  • Consider vibratome sectioning of fresh tissue to minimize fixation artifacts

• Blocking enhancements:

  • Use multi-component blocking solution:

    • 5% normal serum (from secondary antibody host species)

    • 3% BSA

    • 0.3% Triton X-100

    • Optional: 0.1% gelatin for additional blocking

• Antibody incubation optimization:

  • Pre-absorb antibody with plant extract from arf21 mutant

  • Extend primary antibody incubation time (overnight at 4°C) with reduced concentration

  • Increase washing stringency (more washes, higher salt concentration)

• Detection system selection:

  • Use highly cross-adsorbed secondary antibodies

  • Consider tyramide signal amplification for low-abundance targets

  • Employ species-specific secondary antibodies to minimize non-specific binding

• Essential controls:

  • No primary antibody control

  • Isotype control (irrelevant antibody of same isotype)

  • Pre-immune serum control

  • Peptide competition control

  • arf21 mutant tissue control

These strategies collectively minimize non-specific binding while maintaining sensitivity for specific detection of ARF21 protein in complex plant tissue sections.

How should researchers address weak or inconsistent signals when using ARF21 antibody in Western blots?

When facing weak or inconsistent signals, systematic troubleshooting is essential:

For plant-specific issues, consider adding 2% PVPP to extraction buffer to remove phenolic compounds that may interfere with antibody binding. Also, consider using an alternative membrane type (PVDF instead of nitrocellulose) which may provide better protein retention and signal-to-noise ratio for ARF21 detection.

What are the most common pitfalls in co-immunoprecipitation experiments using ARF21 antibody?

Co-immunoprecipitation (co-IP) with ARF21 antibody can face several challenges:

• Extraction buffer limitations:

  • Too harsh: May disrupt protein-protein interactions

  • Too mild: May result in high background from non-specific binding

  • Solution: Test gradient of detergent concentrations (0.1-1% NP-40 or Triton X-100)

• Plant-specific interference:

  • Cell wall components can create non-specific aggregates

  • Solution: Include 0.1% cellulase in extraction buffer or perform protoplast isolation

• Cross-linking considerations:

  • No cross-linking: May lose transient interactions

  • Excessive cross-linking: May capture non-physiological interactions

  • Solution: Optimize formaldehyde concentration (0.1-1%) and cross-linking time

• Antibody orientation issues:

  • Direct binding of antibody to beads may block critical epitopes

  • Solution: Use oriented coupling techniques or commercial kits designed for optimal antibody presentation

• Elution challenges:

  • Harsh elution: May denature interacting proteins

  • Incomplete elution: May lose low-abundance interactors

  • Solution: Compare acidic glycine elution vs. competitive peptide elution vs. SDS elution

• Essential controls often overlooked:

  • IgG control to identify non-specific binding

  • Input sample (5-10%) to verify starting material

  • arf21 mutant extract as negative control

  • Reciprocal IP with antibodies against suspected interacting partners

Addressing these pitfalls systematically increases the likelihood of identifying genuine ARF21 interacting partners while minimizing false positives common in plant co-IP experiments.

How can researchers resolve contradictory results between immunoblotting and immunofluorescence when using ARF21 antibody?

Contradictory results between different techniques require systematic investigation:

• Method-specific factors:

  • Western blot detects denatured proteins; some epitopes may be masked in native conditions

  • Immunofluorescence preserves spatial information but may have fixation artifacts

  • ELISA uses native or partially denatured proteins depending on protocol

• Systematic evaluation approach:

  Technique	Validate By	Critical Controls
  Western blot	Testing multiple extraction methods	Include arf21 mutant extract
  Immunofluorescence	Try different fixation protocols	Include secondary-only control
  ELISA	Test different coating conditions	Include dilution series to confirm linearity

• Biological explanations to consider:

  • Post-translational modifications may affect epitope recognition differently between methods

  • Protein isoforms may be detected differentially by different techniques

  • Protein conformation changes in different subcellular compartments

• Resolution strategies:

  • Use epitope-tagged ARF21 constructs to validate antibody specificity across methods

  • Perform epitope mapping to understand antibody binding determinants

  • Consider generating new antibodies against different regions of ARF21

  • Document conditions under which each result is observed for proper interpretation

When publishing results, transparently report these validation steps and acknowledge method-specific limitations to enhance reproducibility of ARF21 research.

How can ARF21 antibody be effectively utilized in chromatin immunoprecipitation studies?

If ARF21 functions in transcriptional regulation (like some plant ARF family proteins), chromatin immunoprecipitation (ChIP) would be valuable. For effective ChIP using ARF21 antibody:

• Cross-linking optimization:

  • Test formaldehyde concentrations (0.5-2%)

  • Optimize cross-linking time (5-20 minutes)

  • Quench with glycine (125 mM final concentration)

• Chromatin preparation:

  • Sonicate to achieve 200-500 bp DNA fragments

  • Verify fragmentation by agarose gel electrophoresis

  • Pre-clear chromatin with Protein A/G beads and non-immune IgG

• Immunoprecipitation refinement:

  • Test different antibody amounts (2-10 μg per reaction)

  • Extend incubation time (overnight at 4°C with rotation)

  • Perform stringent washing (increasing salt concentrations)

• Plant-specific considerations:

  • Filter through Miracloth to remove plant debris

  • Include PVP (2%) in extraction buffers to remove phenolics

  • Consider specialized plant ChIP protocols (e.g., INTACT method for nuclear isolation)

• Controls and validation:

  • Input DNA (5-10% of starting material)

  • IgG negative control

  • Positive control loci (known ARF-binding sites if available)

  • Negative control loci (inactive genes)

• Data analysis approach:

  • qPCR with primers flanking suspected binding sites

  • Calculate enrichment as percent input or relative to IgG

  • For genome-wide studies, consider ChIP-seq with appropriate bioinformatic analysis

The success of ChIP experiments with ARF21 antibody critically depends on antibody specificity and the abundance of ARF21 at chromatin binding sites.

What advanced protein-protein interaction methods beyond co-IP can be employed with ARF21 antibody?

Several sophisticated techniques can complement traditional co-IP approaches:

• Proximity labeling approaches:

  • BioID methodology:

    • Express ARF21-BirA fusion to biotinylate proximal proteins

    • Use ARF21 antibody to validate fusion protein localization

    • Identify biotinylated proteins via streptavidin pulldown and mass spectrometry

  • APEX/TurboID systems:

    • Faster labeling kinetics than BioID (minutes vs. hours)

    • Better spatial resolution for compartment-specific interactions

    • Can capture transient interactions missed by co-IP

• In situ interaction detection:

  • Proximity Ligation Assay (PLA):

    • Detects protein interactions with spatial resolution in fixed samples

    • Requires ARF21 antibody and antibody against potential interactor

    • Signal amplification enables detection of low-abundance interactions

  • FRET-based approaches:

    • Use ARF21 antibody to validate expression patterns

    • Measure direct protein-protein interactions in living cells

    • Quantify interaction dynamics under different conditions

• Antibody-based protein complex purification:

  • Blue Native PAGE followed by immunodetection

  • Size-exclusion chromatography with antibody detection

  • Density gradient fractionation with ARF21 antibody detection

• Multi-antibody approaches:

  • Sequential IP:

    • First IP with ARF21 antibody

    • Elute and re-IP with antibody against suspected partner

    • Stringently validates direct interactions

These advanced methods provide complementary data to traditional co-IP, offering insights into spatial localization, interaction dynamics, and complex composition that are difficult to obtain with co-IP alone.

How can researchers apply ARF21 antibody in studying post-translational modifications?

Investigating post-translational modifications (PTMs) of ARF21 requires specialized approaches:

• Phosphorylation analysis:

  • Immunoprecipitate using ARF21 antibody

  • Analyze by:

    • Western blot with phospho-specific antibodies

    • Phosphatase treatment to confirm modification

    • Mass spectrometry for site identification

  • Compare phosphorylation status under different conditions

• Ubiquitination detection:

  • Immunoprecipitate with ARF21 antibody under denaturing conditions

  • Probe with anti-ubiquitin antibodies

  • Alternatively, express His-tagged ubiquitin and perform Ni-NTA pulldown

  • Detect ARF21 in pulldown material using ARF21 antibody

• SUMOylation analysis:

  • Similar approach to ubiquitination detection

  • Use SUMO-specific antibodies or tagged SUMO constructs

  • Consider SUMO-specific protease treatment as confirmation

• PTM-focused mass spectrometry:

  • Immunoprecipitate with ARF21 antibody

  • Perform targeted MS analysis for specific modifications

  • Use enrichment strategies for phosphopeptides, ubiquitinated peptides, etc.

  • Quantify changes in modification states across conditions

• Modification-specific interaction studies:

  • Determine if certain ARF21 interactions are modification-dependent

  • Compare interactomes of modified vs. unmodified protein

  • Use phosphomimetic or phospho-dead mutations to validate functional significance

These approaches can reveal how ARF21 activity, localization, and interactions are regulated through post-translational mechanisms, providing deeper insights into its cellular functions.

What cutting-edge imaging techniques can be combined with ARF21 antibody for subcellular localization studies?

Modern imaging approaches can significantly enhance ARF21 localization studies:

• Super-resolution microscopy:

  • Structured Illumination Microscopy (SIM):

    • 2× improvement in resolution over confocal

    • Compatible with standard immunofluorescence protocols

    • Allows visualization of ARF21 in relation to cellular structures

  • Stochastic Optical Reconstruction Microscopy (STORM):

    • ~20 nm resolution potential

    • Requires photoswitchable fluorophores conjugated to secondary antibodies

    • Enables precise mapping of ARF21 distribution patterns

• Expansion microscopy:

  • Physical expansion of immunolabeled samples in hydrogel

  • Achieves effective super-resolution with standard confocal microscopy

  • Particularly valuable for dense plant tissues

• Correlative light and electron microscopy (CLEM):

  • Immunofluorescence with ARF21 antibody followed by electron microscopy

  • Permits ultrastructural context for ARF21 localization

  • Options include pre-embedding or post-embedding immunogold labeling

• Multi-scale imaging approaches:

  • Whole-plant imaging to tissue to cellular to subcellular localization

  • Combines macro imaging with high-resolution techniques

  • Maps ARF21 distribution across biological scales

• Live-cell proximity labeling imaging:

  • Express ARF21 fused to HaloTag or SNAP-tag

  • Validate localization pattern with fixed-cell ARF21 antibody staining

  • Perform pulse-chase labeling to track protein dynamics

• Quantitative analysis frameworks:

  • Automated segmentation of subcellular compartments

  • Colocalization analysis with Pearson's or Mander's coefficients

  • Machine learning classification of distribution patterns

These advanced imaging approaches, when combined with rigorous ARF21 antibody validation, provide unprecedented insights into the spatial organization and dynamics of ARF21 in plant cells.

How can artificial intelligence technologies enhance antibody-based research for plant proteins like ARF21?

Recent advances in AI are transforming antibody research approaches:

• AI-driven antibody development:

  • Machine learning algorithms can predict optimal epitopes for antibody generation

  • Computational modeling can improve antibody specificity and affinity

  • AI systems can design optimal validation experiments based on protein properties

• Advanced image analysis:

  • Deep learning networks can automatically segment and analyze immunofluorescence images

  • Computer vision algorithms can quantify changes in protein distribution patterns

  • AI can detect subtle phenotypes in ARF21 mutant vs. wild-type plants

• Predictive modeling applications:

  • Predict ARF21 interaction partners based on structural properties

  • Model effects of post-translational modifications on ARF21 function

  • Simulate ARF21 binding to potential DNA targets

As highlighted in recent research, Vanderbilt University Medical Center has been awarded $30 million to develop AI technologies for therapeutic antibody discovery . Similar approaches could be applied to plant research antibodies like those targeting ARF21, potentially revolutionizing how researchers design experiments and interpret results.

What novel research applications might emerge from combining ARF21 antibody with molecular fate-mapping approaches?

Molecular fate-mapping, a technique recently developed for tracking antibody origins , could be adapted for plant research with ARF21:

• Developmental tracking:

  • Monitor ARF21 expression across developmental stages

  • Identify cell lineages where ARF21 plays critical roles

  • Distinguish between different ARF21 protein populations (newly synthesized vs. persistent)

• Stress response dynamics:

  • Track ARF21 fate during environmental stress responses

  • Monitor protein turnover rates under different conditions

  • Identify stress-specific post-translational modifications

• Protein lifetime studies:

  • Pulse-chase experiments with tagged ARF21

  • Validation with antibodies against endogenous protein

  • Quantify half-life in different tissues and conditions

• Single-cell applications:

  • Combine with single-cell transcriptomics

  • Correlate ARF21 protein fate with gene expression patterns

  • Identify cell-specific regulation mechanisms

The molecular fate-mapping approach demonstrated for serum antibodies could be adapted to study intracellular protein dynamics, providing new insights into ARF21 regulation and function in plant development and stress responses.