At4g12090 Antibody

What is At4g12090 and why is it significant in plant research?

At4g12090 encodes Protein cornichon homolog 5 in Arabidopsis thaliana, a 135 amino acid protein with the sequence MGDLLDWIISFLFLATLIIIVIYQLTCLADLEFDRINPYDVSSRINRMVLPEFGLQGLLCLYYILTGHWFMAVLSLPHLFYNIRLYMKREHLADVTELYNTNKWEQKKRVYKIGHIALSIFITTYWLIHSALGDI . This protein belongs to the cornichon protein family, which plays roles in protein trafficking and secretory pathways in plants. Understanding its function requires specific and validated antibodies to detect its expression, localization, and interactions with other cellular components. The protein's relatively small size makes antibody selection particularly important for accurate detection and characterization.

What types of At4g12090 antibodies are commercially available?

Researchers can access several types of antibodies targeting different regions of the At4g12090 protein. These typically include:

• N-terminal specific antibodies (e.g., X-Q9SZ74-N): These target the amino terminus of the protein and consist of combinations of monoclonal antibodies against synthetic peptides representing the N-terminal sequence .

• C-terminal specific antibodies (e.g., X-Q9SZ74-C): These target the carboxyl terminus and are also available as combinations of monoclonal antibodies .

• Middle region antibodies (e.g., X-Q9SZ74-M): These target non-terminus sequences and provide an alternative epitope recognition pattern .

Each antibody type offers different advantages depending on the experimental goals and the structural accessibility of protein domains in various applications.

How should researchers select between different At4g12090 antibody options?

Selection of the appropriate antibody should be based on:

• Experimental application (Western blot, immunoprecipitation, immunohistochemistry)

• Protein conformation in the experimental conditions

• Potential cross-reactivity with related proteins

• Published validation data for specific applications

For membrane proteins like cornichon homologs, antibodies targeting extracellular domains may be preferable for certain applications, while antibodies targeting cytoplasmic domains may be better for others. Always review the ELISA titer information, which for most At4g12090 antibodies is approximately 10,000, corresponding to detection sensitivity of around 1 ng of target protein on Western blots .

What validation steps are essential before using At4g12090 antibodies?

Thorough validation of antibodies is critical for generating reliable research data. For At4g12090 antibodies, researchers should:

• Test for specificity using positive and negative controls (wild-type vs. knockout plants)

• Evaluate sensitivity through dilution series experiments

• Assess reproducibility across multiple experimental replicates

• Verify cross-reactivity with related cornichon family proteins

• Validate in the specific experimental conditions to be used in the study

Signal-to-noise ratio and dynamic range are particularly important objective parameters when validating antibodies for quantitative protein evaluation. Using too high an antibody concentration can yield nonspecific results, while too low a concentration can lead to false-negative results .

How can researchers determine the optimal working concentration for At4g12090 antibodies?

Optimal antibody concentration determination requires:

• Performing titration experiments with decreasing antibody concentrations (typically starting at 1:500 and diluting to 1:10,000)

• Evaluating signal-to-noise ratio at each concentration

• Identifying the concentration that provides maximum specific signal with minimal background

• Testing across different sample preparations to ensure consistency

For At4g12090 antibodies with ELISA titers around 10,000, starting dilutions of 1:1,000 to 1:2,000 are typically appropriate for Western blotting applications, but optimization is essential for each specific experimental system .

What controls are essential when using At4g12090 antibodies?

Rigorous experimental controls include:

What are the optimal protein extraction methods for At4g12090 detection?

As a membrane-associated protein, At4g12090 requires careful extraction methods:

• Use detergent-based extraction buffers containing mild non-ionic detergents (0.5-1% Triton X-100 or NP-40)

• Include protease inhibitors to prevent degradation during extraction

• Optimize buffer pH (typically 7.4-8.0) and ionic strength

• Consider membrane fractionation techniques to enrich for membrane-associated proteins

• Avoid harsh denaturants that might disrupt epitope recognition unless required for specific applications

Temperature control during extraction is particularly important, with all steps performed at 4°C to minimize protein degradation and maintain epitope integrity for subsequent antibody recognition.

What are the recommended protocols for Western blotting with At4g12090 antibodies?

Optimal Western blotting protocols include:

• Sample preparation: Use SDS-PAGE sample buffer with reducing agents for most applications

• Gel percentage: 12-15% acrylamide gels are appropriate for the 135 AA At4g12090 protein

• Transfer conditions: Semi-dry or wet transfer to PVDF membranes (preferred over nitrocellulose for small proteins)

• Blocking solution: 5% non-fat dry milk or 3% BSA in TBST (Tris-buffered saline with 0.1% Tween-20)

• Primary antibody incubation: Overnight at 4°C at optimized dilution (typically 1:1,000 to 1:5,000)

• Secondary antibody: HRP-conjugated anti-mouse IgG at 1:5,000 to 1:10,000 dilution

• Detection method: Enhanced chemiluminescence with appropriate exposure times

For quantitative analysis, consider using fluorescently-labeled secondary antibodies and detection systems that provide wider dynamic range than chemiluminescence.

What are effective antigen retrieval methods for immunohistochemistry applications?

For immunohistochemical detection of At4g12090:

• Start with vendor-recommended retrieval methods, as these have likely been optimized for the specific antibody

• Common methods include:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Enzymatic retrieval using proteinase K (1-5 μg/ml for 10-20 minutes)

  • Tris-EDTA buffer (pH 9.0) for certain membrane proteins

• Optimization may require testing multiple retrieval methods and conditions

• After changing retrieval methods, re-optimize antibody concentration, as epitope accessibility may change

Note that as antigen retrieval methods are altered, the optimal antibody concentration might need adjustment as well to maintain specificity and sensitivity.

What are common causes of non-specific binding when using At4g12090 antibodies?

Non-specific binding issues can arise from several sources:

• Insufficient blocking: Increase blocking time or concentration, or try alternative blocking agents

• Excessive antibody concentration: Dilute primary antibody further

• Cross-reactivity with related proteins: Use peptide competition assays to confirm specificity

• Sample preparation issues: Ensure complete denaturation for Western blots or proper fixation for immunohistochemistry

• Secondary antibody background: Include secondary-only controls and consider alternative secondary antibodies

For At4g12090 antibodies classified as "Crazy" in the AbClass™ system, particular attention to optimization is required as this classification often indicates challenging antibody behavior that may require more extensive validation and protocol refinement .

How can researchers address inconsistent results with At4g12090 antibodies?

Inconsistency in antibody performance can be addressed through:

• Standardization of protocols and reagent preparation

• Careful control of environmental factors (temperature, humidity, incubation times)

• Aliquoting antibodies to avoid freeze-thaw cycles

• Preparing fresh working solutions for each experiment

• Implementing detailed record-keeping of lot numbers and experimental conditions

When inconsistencies persist, consider testing different epitope-targeting antibodies (N-terminal vs. C-terminal) as protein modifications or degradation may affect epitope availability in different experimental contexts .

What are advanced approaches for studying At4g12090 protein interactions?

Advanced techniques for studying At4g12090 interactions include:

• Co-immunoprecipitation using At4g12090 antibodies followed by mass spectrometry

• Proximity labeling approaches such as BioID or APEX2 fused to At4g12090

• Fluorescence resonance energy transfer (FRET) between At4g12090 and potential interacting partners

• Split-GFP complementation assays in planta

• Yeast two-hybrid screening using At4g12090 as bait

When studying receptor-mediated processes similar to those observed in other systems, researchers might consider approaches used in receptor-mediated transcytosis studies that carefully track antibody internalization and trafficking through specific cellular compartments .

How can researchers effectively monitor antibody internalization and trafficking in At4g12090 studies?

For researchers interested in monitoring At4g12090 antibody internalization and trafficking:

• Use fluorescently-conjugated antibodies (similar to Alexa Fluor® 647-conjugated antibodies used in other receptor studies)

• Implement live-cell imaging with appropriate markers for different endosomal compartments

• Consider pulse-chase experiments to track antibody movement through cellular compartments

• Use endosomal markers (EEA1 for early endosomes, Rab7 for late endosomes) to characterize trafficking pathways

• Implement super-resolution microscopy for detailed co-localization studies

Methods developed for studying sorting receptors like transferrin receptor and sortilin could be adapted for At4g12090 studies, particularly if this protein plays a role in cellular trafficking pathways .

What are best practices for quantifying At4g12090 expression levels?

Accurate quantification requires:

• Use of housekeeping gene/protein controls appropriate for the experimental conditions

• Application of digital image analysis software with appropriate background subtraction

• Establishment of standard curves using purified recombinant proteins when available

• Inclusion of biological and technical replicates (minimum n=3)

• Statistical analysis appropriate for the experimental design and data distribution

When quantifying signal from Western blots, ensure measurements are taken in the linear range of detection to avoid saturation effects that can lead to underestimation of differences between samples.

How should researchers report At4g12090 antibody usage in publications?

Complete antibody reporting should include:

• Full antibody identification (catalog number, lot number, manufacturer)

• Validation performed specifically for the reported application

• Detailed methodological information (concentrations, incubation times, buffers)

• All control experiments conducted

• Unmodified representative images with scale bars

This comprehensive reporting is essential for research reproducibility and allows other researchers to accurately build upon published findings .

What emerging technologies might improve At4g12090 detection sensitivity?

Emerging technologies include:

• Single-molecule detection methods for low-abundance proteins

• Automated Western blot systems with higher reproducibility

• Multiplexed detection systems for simultaneous analysis of multiple proteins

• Capillary-based immunoassays with higher sensitivity than traditional Western blots

• Advanced cryo-electron microscopy for structural analysis of antibody-antigen complexes

These approaches may be particularly valuable for studying low-abundance membrane proteins like At4g12090, especially in specific cellular compartments or developmental stages where expression levels may be minimal.