At3g18340 Antibody

What is the At3g18340 protein and why is it studied in plant research?

At3g18340 is a protein encoded by the Arabidopsis thaliana genome (accession Q9LS56). It is studied in plant research to understand cellular structures and protein functions during flower development, which is one of the most complex structures of angiosperms essential for sexual reproduction. The antibody against this protein serves as a molecular marker for investigating biological mechanisms underlying floral development in plants. Arabidopsis thaliana (mouse-ear cress) is a model organism widely used in plant molecular biology due to its small genome size, rapid life cycle, and genetic tractability .

What are the standard applications for At3g18340 antibody?

The standard applications for At3g18340 antibody include:

• Western Blot (WB): Used to detect and quantify the At3g18340 protein in tissue extracts

• Enzyme-Linked Immunosorbent Assay (ELISA): Used for sensitive detection of the protein in solution

• Immunofluorescence microscopy: Used to visualize protein localization in tissue sections

• Immunoprecipitation: Used to isolate and purify the At3g18340 protein or protein complexes

The antibody has been specifically tested for ELISA and Western Blot applications to ensure identification of the antigen . The characterization of antibodies through multiple techniques is important for establishing their reliability as research tools in plant biology studies .

What is the optimal storage protocol for At3g18340 antibody?

The optimal storage protocol for At3g18340 antibody is as follows:

• Upon receipt, store at -20°C or -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles as this can degrade the antibody and reduce its effectiveness

• The antibody is typically supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• For working aliquots, small volumes can be prepared and stored at 4°C for short-term use (1-2 weeks)

Proper storage is critical to maintain antibody functionality and specificity for experimental applications . The presence of glycerol in the storage buffer helps prevent freeze damage during storage at subzero temperatures.

How should I validate the specificity of At3g18340 antibody in my experimental system?

Validating the specificity of At3g18340 antibody should follow a multi-step approach:

• Positive and negative controls: Include samples from wildtype Arabidopsis and knockout/knockdown lines of At3g18340 if available

• Cross-reactivity testing: Test the antibody on proteins from related species to assess specificity

• Peptide competition assay: Pre-incubate the antibody with purified At3g18340 protein or peptide before application to confirm specific binding

• Multiple detection methods: Validate results using both Western blot and immunofluorescence to confirm consistent detection patterns

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the detected protein

Research has shown that antibody validation is critical for ensuring reproducible results in plant biology. Strategies for monoclonal antibody screening developed for Arabidopsis can be adapted to validate polyclonal antibodies like At3g18340 . Importantly, validation should be performed in each experimental system as tissue-specific factors may affect antibody performance.

What are the optimal Western blot conditions for At3g18340 antibody?

The optimal Western blot conditions for At3g18340 antibody typically include:

• Sample preparation:

  • Extract proteins from Arabidopsis tissues using a buffer containing protease inhibitors

  • Determine protein concentration using Bradford or BCA assay

  • Use 20-50 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Dilute primary At3g18340 antibody 1:1000 to 1:2000 in blocking buffer

  • Incubate membrane with primary antibody overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Wash 3-5 times with TBST

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Expose to X-ray film or use imaging system with appropriate exposure times

Western blot optimization may be necessary for each specific application, as indicated by studies on antibody performance in plant tissues . Consistent technique is crucial for reliable detection of the At3g18340 protein.

What protocol should be followed for immunofluorescence microscopy with At3g18340 antibody?

For immunofluorescence microscopy with At3g18340 antibody, follow this protocol:

• Tissue preparation:

  • Fix Arabidopsis inflorescence tissues in 4% paraformaldehyde

  • Dehydrate through an ethanol series

  • Embed in paraffin

  • Section tissues to 8-10 μm thickness

  • Mount on adhesive slides

• Antigen retrieval and blocking:

  • Deparaffinize sections with xylene and rehydrate through decreasing ethanol series

  • Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

  • Block with 2-5% BSA or normal serum in PBS for 1 hour at room temperature

• Antibody incubation:

  • Dilute At3g18340 antibody 1:100 to 1:500 in blocking buffer

  • Incubate sections overnight at 4°C in a humid chamber

  • Wash 3 times with PBS

  • Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488) at 1:500 dilution for 1-2 hours at room temperature

  • Wash 3 times with PBS

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

• Imaging:

  • Use confocal or fluorescence microscope

  • Include appropriate positive and negative controls

  • Capture images at multiple magnifications to visualize cellular and subcellular localization

This protocol is based on established immunofluorescence methods used for Arabidopsis floral tissues, which have successfully revealed distinct cellular distribution patterns of epitopes in flower sections .

What are the most common reasons for false-negative results with At3g18340 antibody?

Several factors can contribute to false-negative results when using At3g18340 antibody:

• Protein degradation: Inadequate protease inhibitors or improper sample handling can lead to protein degradation

• Insufficient antigen retrieval: For fixed tissues, incomplete antigen retrieval can mask epitopes

• Inappropriate fixation: Overfixation can cross-link proteins and mask epitopes

• Antibody degradation: Improper storage or repeated freeze-thaw cycles can reduce antibody activity

• Low expression levels: The target protein may be expressed at levels below detection threshold

• Post-translational modifications: Modifications of the protein may alter epitope recognition

• Tissue-specific expression: The At3g18340 protein might show tissue-specific expression patterns and may not be present in all plant tissues

To address these issues, researchers should optimize protein extraction protocols, try different antigen retrieval methods, and test antibody activity with positive control samples. Studies on antibody characterization in plant tissues have demonstrated that optimization is often necessary for each specific application .

How can I determine the detection sensitivity of At3g18340 antibody?

To determine the detection sensitivity of At3g18340 antibody:

• Standard curve generation:

  • Prepare serial dilutions of recombinant At3g18340 protein (if available)

  • Perform Western blot or ELISA with the dilution series

  • Plot signal intensity versus protein concentration

  • Determine the limit of detection (LOD) - typically the concentration giving a signal 3x above background

• Titration experiments:

  • Prepare various dilutions of the antibody (1:500, 1:1000, 1:2000, etc.)

  • Test each dilution on the same amount of sample

  • Determine the optimal antibody concentration that provides specific signal with minimal background

• Comparison with quantified samples:

  • Use samples with known quantities of the target protein

  • Compare signal intensities to establish a standard curve

  • Use this curve to estimate unknown concentrations in experimental samples

• Image analysis software:

  • Use densitometry software for Western blot quantification

  • Establish signal-to-noise ratios to determine sensitivity limits

A methodical approach to sensitivity determination helps ensure reliable experimental results and appropriate antibody usage across different applications .

What strategies exist for optimizing immunoprecipitation with At3g18340 antibody?

Optimizing immunoprecipitation with At3g18340 antibody involves several strategies:

• Sample preparation optimization:

  • Test different lysis buffers (varying salt concentrations, detergents, and pH)

  • Include protease and phosphatase inhibitors

  • Optimize protein extraction conditions for plant tissues (e.g., grinding in liquid nitrogen)

• Antibody coupling methods:

  • Direct coupling to beads (e.g., cyanogen bromide-activated Sepharose)

  • Protein A/G beads for immune complex capture

  • Magnetic beads for gentler handling

• Binding conditions optimization:

  • Adjust antibody-to-sample ratio

  • Optimize incubation time (4 hours to overnight)

  • Test different temperatures (4°C is typically preferred)

• Washing stringency adjustment:

  • Modify salt concentration in wash buffers

  • Adjust detergent type and concentration

  • Increase or decrease number of washes

• Elution method selection:

  • pH elution (glycine buffer pH 2.5-3.0)

  • Competitive elution with antigenic peptide

  • SDS and heat for complete elution

• Confirmation by mass spectrometry:

  • Perform LC-MS/MS analysis of immunoprecipitated proteins

  • Confirm target and identify interaction partners

Studies have shown that successful immunoprecipitation followed by mass spectrometry analysis can effectively identify potential targets for antibodies and their interacting partners .

How does At3g18340 antibody compare to other antibodies for Arabidopsis research?

The At3g18340 antibody presents specific characteristics that differentiate it from other Arabidopsis research antibodies:

Unlike commercial antibodies against known antigens such as GFP, YFP, and FLAG that are commonly used for tagged proteins, the At3g18340 antibody enables direct study of the native protein. This is particularly valuable for understanding natural protein expression patterns and interactions without potential artifacts introduced by protein tagging .

Current studies using molecular genetic tools have made great advances in understanding flower development, but knowledge about cellular structures in floral organs has been limited due to the scarcity of antibodies that can label cellular components. The At3g18340 antibody contributes to addressing this limitation .

What experimental approaches can combine At3g18340 antibody with genetic studies in Arabidopsis?

Several experimental approaches can integrate At3g18340 antibody with genetic studies:

• Mutant phenotype correlation:

  • Compare At3g18340 protein levels in wildtype and mutant backgrounds

  • Correlate protein expression with developmental phenotypes

  • Analyze protein localization changes in different genetic backgrounds

• Protein-protein interaction networks:

  • Use At3g18340 antibody for co-immunoprecipitation studies

  • Identify interaction partners through mass spectrometry

  • Validate interactions using reciprocal co-IP or yeast two-hybrid assays

• Chromatin immunoprecipitation (ChIP):

  • If At3g18340 is a DNA-binding protein, use the antibody for ChIP experiments

  • Identify genomic binding sites through ChIP-seq

  • Correlate binding with gene expression changes

• Tissue-specific expression analysis:

  • Compare protein localization with promoter-reporter gene studies

  • Analyze protein expression in various tissues and developmental stages

  • Correlate with RNA-seq or microarray data

• Functional complementation:

  • Express wildtype or mutant versions of At3g18340 in knockout lines

  • Use the antibody to verify protein expression levels

  • Correlate protein expression with phenotypic rescue

These integrated approaches leverage the strengths of both antibody-based protein studies and genetic analyses to provide more comprehensive insights into protein function .

How can At3g18340 antibody be used in multiplex immunofluorescence imaging?

At3g18340 antibody can be effectively used in multiplex immunofluorescence imaging through these approaches:

• Sequential staining protocol:

  • Perform serial rounds of antibody labeling, imaging, and signal quenching

  • Use different fluorophore-conjugated secondary antibodies for distinct targets

  • Ensure complete signal removal between rounds to prevent cross-detection

• Spectral unmixing techniques:

  • Simultaneously use multiple primary antibodies from different host species

  • Apply spectrally distinct secondary antibodies

  • Use confocal microscopy with spectral detection and linear unmixing algorithms

• Tyramide signal amplification (TSA):

  • Enhance signal detection sensitivity for low-abundance proteins

  • Allow the use of multiple primary antibodies from the same host species

  • Enable serial detection of multiple targets through sequential TSA reactions

• Integration with the IBEX multiplex tissue imaging platform:

  • Combine At3g18340 antibody with other validated antibodies in the IBEX system

  • Apply iterative staining, imaging, and signal removal

  • Create highly multiplexed images of plant tissue architecture

• Colocalization analysis:

  • Combine At3g18340 antibody with antibodies against known cellular markers

  • Determine subcellular localization through quantitative colocalization analysis

  • Calculate Pearson's or Mander's coefficients to quantify colocalization

Multiplex immunofluorescence imaging has been successfully applied in tissue imaging repositories and can significantly enhance the understanding of protein spatial relationships in plant tissues .

What emerging technologies might enhance research applications of At3g18340 antibody?

Several emerging technologies show promise for enhancing At3g18340 antibody applications:

• Super-resolution microscopy techniques:

  • Structured Illumination Microscopy (SIM) provides 2x conventional resolution

  • Stimulated Emission Depletion (STED) microscopy enhances resolution to ~50 nm

  • Single-Molecule Localization Microscopy (PALM/STORM) achieves 20-30 nm resolution

  • These techniques can reveal detailed subcellular localization beyond diffraction limits

• Proximity labeling approaches:

  • Combining At3g18340 antibody with BioID or APEX2 proximity labeling

  • Identifying proteins in close proximity to At3g18340 in living cells

  • Creating spatial protein interaction maps in specific tissues

• Live-cell antibody applications:

  • Development of cell-penetrating antibody fragments based on 3E10 framework

  • Real-time monitoring of protein dynamics in living plant cells

  • Targeted protein modulation through antibody-based inhibition

• Antibody engineering:

  • Creating single-chain variable fragments (scFvs) for improved tissue penetration

  • Developing recombinant antibodies with enhanced specificity and affinity

  • Engineering nanobodies (VHH antibodies) for specialized applications

• Automated high-throughput screening:

  • Robotics-assisted antibody validation and characterization

  • Large-scale screening of antibody specificity across multiple conditions

  • Integration with machine learning for improved antibody applications

These technologies build upon recent advancements in cell-penetrating monoclonal antibody therapies and new genotype-phenotype linked antibody development methods, which could be adapted for plant research applications .

How can I develop a quantitative assay using At3g18340 antibody for protein expression analysis?

Developing a quantitative assay using At3g18340 antibody involves these key steps:

• Quantitative Western blot development:

  • Generate a standard curve using purified recombinant At3g18340 protein

  • Use fluorescent or chemiluminescent detection with linear dynamic range

  • Include loading controls (e.g., actin, tubulin) for normalization

  • Apply image analysis software for accurate densitometry

• Sandwich ELISA protocol:

  • Coat plates with a capture antibody (At3g18340 or another antibody against the same protein)

  • Apply samples and standards with known protein concentrations

  • Detect using At3g18340 antibody followed by enzyme-conjugated secondary antibody

  • Generate a standard curve for quantification of unknown samples

• Implementing digital ELISA technologies:

  • Adapt single-molecule array (Simoa) techniques for ultra-sensitive detection

  • Use paramagnetic beads conjugated with At3g18340 antibody

  • Apply digital counting of individual enzyme-substrate reactions for quantification

• Flow cytometry application:

  • Fix and permeabilize plant protoplasts

  • Stain with At3g18340 antibody and fluorophore-conjugated secondary antibody

  • Analyze fluorescence intensity as a measure of protein expression

  • Sort cells based on expression levels if needed

• Image-based quantification:

  • Perform immunofluorescence staining with At3g18340 antibody

  • Capture images using identical acquisition parameters

  • Quantify signal intensity using image analysis software

  • Normalize to cell number or tissue area

These quantitative approaches build upon established protocols for antibody-based protein detection while incorporating advanced techniques for accurate quantification .

What considerations are important when using At3g18340 antibody for evolutionary studies across plant species?

When using At3g18340 antibody for evolutionary studies across plant species, several considerations are crucial:

• Epitope conservation assessment:

  • Perform sequence alignment of At3g18340 orthologs across target species

  • Identify conserved and variable regions that might affect antibody binding

  • Predict epitope conservation using bioinformatics tools

• Cross-reactivity validation:

  • Test antibody on protein extracts from multiple species

  • Verify specific binding through Western blot analysis

  • Confirm consistent molecular weight detection accounting for species variations

• Sensitivity calibration:

  • Determine detection sensitivity in each species

  • Account for potential affinity differences due to protein sequence variations

  • Adjust antibody concentrations accordingly for comparable results

• Tissue-specific expression patterns:

  • Compare protein localization across homologous tissues in different species

  • Document conserved and divergent expression patterns

  • Correlate with developmental stages for proper comparisons

• Integration with phylogenetic analysis:

  • Map protein expression/localization data onto phylogenetic trees

  • Identify evolutionary patterns in protein function and regulation

  • Correlate with genomic changes across the phylogeny

• Controls and validation:

  • Include positive controls from Arabidopsis thaliana

  • Use antibodies against highly conserved proteins as internal controls

  • Consider negative controls using pre-immune serum

This integrated approach can provide valuable insights into the evolution of protein function across plant species while accounting for the technical limitations of antibody cross-reactivity .