ARF14 Antibody

What are ARF14 and ARL14 antibodies, and what biological systems do they target?

ARF14 and ARL14 antibodies target distinct proteins despite their similar nomenclature. ARF14 antibody (e.g., orb784710) targets Auxin Response Factor 14 in Arabidopsis thaliana, a plant transcription factor involved in auxin-responsive gene regulation . In contrast, ARL14 antibody (also known as ARF7 antibody, e.g., ab97811) recognizes ADP-ribosylation factor-like protein 14 in mammals, a GTPase that recruits MYO1E to MHC class II-containing vesicles via the effector protein ARL14EP and controls vesicle movement along the actin cytoskeleton in dendritic cells .

The distinction between these two antibodies is critical for experimental design. ARF14 antibody is plant-specific (Arabidopsis thaliana), while ARL14/ARF7 antibody is designed for mammalian systems, particularly human and mouse samples .

What applications are these antibodies validated for?

The applications for these antibodies differ based on their target systems:

ARF14 Antibody (Plant, orb784710):

• Validated for ELISA and Western Blot (WB) applications

• Specifically designed for plant research in Arabidopsis thaliana

• Unconjugated format allows flexibility in detection systems

ARL14/ARF7 Antibody (Mammalian, ab97811):

• Validated for Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Tested specifically on mouse tissues

• Particularly effective in duodenum tissue analysis

It's important to note that each antibody's validated applications should not be extrapolated to the other, as they target entirely different proteins in different biological systems.

What are the optimal storage and handling conditions to maintain antibody integrity?

For both antibodies, proper storage is critical to maintain functionality. The ARF14 antibody (orb784710) should be stored at 2-8°C for up to 2 weeks for ongoing experiments. For long-term storage, maintain at -20°C in small aliquots to prevent freeze-thaw cycles which can degrade antibody performance . The solution contains 0.03% Proclin 300 and 50% Glycerol as preservatives .

For ARL14/ARF7 antibody (ab97811), similar storage principles apply, though specific preservative formulations may vary between manufacturers. Generally, antibodies benefit from minimizing freeze-thaw cycles by preparing single-use aliquots when storing at -20°C .

What are the recommended dilutions and experimental conditions for optimal results?

For ARL14/ARF7 Antibody (ab97811) in IHC-P:

• Recommended dilution: 1/500

• Antigen retrieval: Citrate buffer, pH 6.0, heat-mediated, 15 minutes

• Tissue types successfully tested: Mouse duodenum tissue

For ARF14 Antibody (orb784710) in Western Blotting:

• Optimal dilutions should be determined experimentally for each lot

• Expected molecular weight for Arabidopsis thaliana ARF14: ~75-80 kDa

• Recommended blocking: 5% non-fat milk or BSA in TBST

These parameters should be considered starting points for optimization. Each laboratory should confirm optimal conditions through careful titration experiments.

How should I design positive and negative controls for validating antibody specificity?

For reliable antibody validation, incorporate these controls:

Positive Controls:

• For ARL14/ARF7: Mouse duodenum tissue sections (known to express ARL14)

• For ARF14: Arabidopsis thaliana wild-type leaf or seedling extracts (expressing ARF14)

Negative Controls:

• Primary antibody omission: Replace primary antibody with same species IgG

• Blocking peptide competition: Pre-incubate antibody with immunizing peptide

• Genetic knockouts/knockdowns: When available, test tissues/cells with target protein deletion or reduction

Validation Methods:

• For ARL14/ARF7: Compare staining patterns with published literature on dendritic cell distribution

• For ARF14: Confirm band size in Western blots matches predicted molecular weight

• Consider orthogonal methods (e.g., mass spectrometry) to confirm protein identity

How can I optimize antigen retrieval for improved ARL14/ARF7 signal in IHC-P?

• Extend retrieval time to 20-30 minutes while maintaining buffer pH

• Test alternative retrieval buffers such as EDTA (pH 8.0) or Tris-EDTA (pH 9.0)

• Explore retrieval methods: microwave, pressure cooker, or water bath

• Fresh tissue fixation: Limit fixation time to 24 hours with 10% neutral buffered formalin

When optimizing, process multiple sections in parallel with systematic modifications to identify optimal conditions. Document all parameters (temperature, duration, buffer composition) for reproducibility.

What is the functional significance of ARL14/ARF7 in dendritic cell biology and how can this antibody advance immunological research?

ARL14/ARF7 functions as a GTPase that recruits MYO1E to MHC class II-containing vesicles through the effector protein ARL14EP, controlling vesicle movement along the actin cytoskeleton in dendritic cells . This mechanism is fundamental to antigen presentation and adaptive immune responses.

Research applications where ARL14/ARF7 antibodies provide significant value include:

• Vesicular Trafficking Studies: Visualizing MHC II-containing compartments in dendritic cells during maturation

• Immune Cell Activation: Tracking changes in ARL14 localization during dendritic cell activation

• Infection Models: Examining pathogen effects on antigen-presentation machinery

• Co-localization Analysis: Combined with markers for endosomal/lysosomal compartments to map trafficking pathways

Researchers should consider dual immunostaining with MHC class II antibodies to assess co-localization and functional relationship in various dendritic cell subsets and activation states.

What approaches are recommended for quantifying ARF14 expression in Arabidopsis using antibody-based methods?

For quantitative analysis of ARF14 expression in Arabidopsis:

Western Blot Densitometry:

• Use standardized protein extraction methods (e.g., TCA-acetone precipitation)

• Load equal amounts of total protein (15-20 μg per lane)

• Include housekeeping controls (e.g., actin, tubulin)

• Use software like ImageJ for density quantification

• Normalize ARF14 signal to loading control

ELISA-Based Quantification:

• Develop a standard curve using recombinant ARF14 protein

• Process plant samples consistently (tissue type, developmental stage)

• Account for extraction efficiency using spike-recovery tests

• Express results as ng ARF14/mg total protein

Tissue-Specific Expression Analysis:

• Compare ARF14 levels across different tissues and developmental stages

• Correlate protein expression with transcript levels (qRT-PCR)

• Consider auxin treatment effects on ARF14 expression and localization

How can I address non-specific binding or high background issues when using these antibodies?

For reducing non-specific binding and background:

In Western Blotting (ARF14 antibody):

• Increase blocking stringency (5% BSA instead of milk, or vice versa)

• Optimize primary antibody concentration through titration experiments

• Increase washing duration/frequency (4-5 times, 10 minutes each)

• Add 0.1-0.5% Triton X-100 or Tween-20 to washing buffer

• Consider alternative blocking agents (e.g., fish gelatin) if plant-specific compounds interfere

In IHC-P (ARL14/ARF7 antibody):

• Use appropriate blocking serum matching secondary antibody species

• Pre-absorb secondary antibodies against tissue powder

• Quench endogenous peroxidases (3% H₂O₂, 10 minutes)

• Block endogenous biotin if using biotin-streptavidin systems

• Reduce antibody concentration while extending incubation time (e.g., overnight at 4°C)

What are the critical considerations when designing multiplexed experiments with ARF14/ARL14 antibodies?

When designing multiplexed experiments:

Panel Design Considerations:

• Antibody species compatibility: Choose primary antibodies from different host species

• Fluorophore selection: Ensure minimal spectral overlap between fluorescent labels

• Expression level balance: Pair highly and lowly expressed targets with appropriate fluorophores

• Antibody cross-reactivity: Test each antibody individually before combining

For ARL14/ARF7 in Multiplex Immunofluorescence:

• Consider sequential staining if using multiple rabbit antibodies

• Test tyramide signal amplification for weak signals

• Validate with single-color controls and fluorescence minus one (FMO) controls

• Use spectral imaging and unmixing for challenging combinations

For ARF14 in Plant Multiplex Studies:

• Consider tissue clearing techniques to improve antibody penetration

• Control for plant autofluorescence (particularly chlorophyll)

• Validate antibody performance on fixed versus fresh tissues

How do post-translational modifications of ARL14/ARF7 potentially affect antibody binding and experimental interpretation?

ARL14/ARF7, being a GTPase, likely undergoes conformational changes between active (GTP-bound) and inactive (GDP-bound) states, which may affect epitope accessibility and antibody binding. Additionally, potential post-translational modifications that may influence antibody detection include:

• GTP/GDP binding status: May alter protein conformation and epitope availability

• Phosphorylation: Could affect antibody recognition, especially for antibodies raised against unmodified peptides

• Lipid modifications: Potential prenylation or myristoylation may affect protein localization and extraction efficiency

• Protein-protein interactions: Binding to effector proteins like ARL14EP may mask antibody epitopes

Researchers should consider these factors when interpreting unexpected results, such as discrepancies between protein levels detected by antibody-based methods versus transcript levels or unexpected subcellular localization patterns.

What emerging research areas might benefit from ARF14/ARL14 antibody applications?

For ARL14/ARF7 in Immunological Research:

• Cancer Immunotherapy: Understanding dendritic cell antigen presentation mechanisms

• Autoimmune Disease Models: Exploring MHC class II trafficking defects

• Infectious Disease Research: Examining pathogen interference with antigen presentation

• Single-Cell Analysis: Combining with mass cytometry for high-dimensional analysis

• Extracellular Vesicle Studies: Investigating ARF7's potential role in exosome formation

For Plant ARF14 in Agricultural Research:

• Stress Response Studies: Examining ARF14's role in auxin-mediated stress adaptations

• Crop Improvement: Understanding auxin signaling networks for enhanced growth

• Root Development: Investigating ARF14's potential role in root architecture

• Hormone Crosstalk: Exploring interactions between auxin and other plant hormones

These emerging areas represent opportunities for researchers to apply ARF14/ARL14 antibodies in novel ways that advance understanding of fundamental biological processes.