At2g29860 Antibody

What is At2g29860 and why is it significant in Arabidopsis research?

At2g29860 is a protein-coding gene in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology. The protein encoded by this gene (UniProt accession O82370) is the target of the At2g29860 Antibody . While the specific function of At2g29860 is not explicitly detailed in the available data, antibodies targeting this protein are valuable tools for investigating protein expression, localization, and function in plant developmental and physiological studies.

When designing experiments with At2g29860 Antibody, researchers should consider:

• The developmental stage of Arabidopsis samples

• Environmental conditions that may affect protein expression

• Tissue-specific expression patterns

• Potential cross-reactivity with related proteins

What are the critical specifications of commercially available At2g29860 Antibody?

The At2g29860 Antibody (e.g., CSB-PA525761XA01DOA) is a polyclonal antibody raised in rabbit against recombinant Arabidopsis thaliana At2g29860 protein . This antibody has been specifically developed for research applications and demonstrates the following specifications:

The antibody is supplied in liquid form and preserved with 0.03% Proclin 300 in a buffer consisting of 50% Glycerol and 0.01M PBS at pH 7.4 . Understanding these specifications is essential for designing appropriate experimental controls and optimizing assay conditions.

What are the recommended storage and handling protocols for At2g29860 Antibody?

Proper storage and handling of At2g29860 Antibody are critical for maintaining its activity and specificity. The recommended storage conditions are:

• Store upon receipt at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as these can compromise antibody activity

• For short-term use, aliquot the antibody and store at 4°C for up to one week

• When working with the antibody, keep it on ice to preserve its activity

For optimal performance, researchers should:

• Aliquot the antibody into small volumes immediately upon receipt

• Use sterile techniques when handling the antibody

• Centrifuge briefly before opening the vial to collect liquid at the bottom

• Document lot numbers and expiration dates for experimental reproducibility

How should I optimize Western blot protocols using At2g29860 Antibody?

Optimizing Western blot protocols for At2g29860 Antibody requires careful consideration of several parameters to ensure specific detection and minimize background. Based on methodologies used for similar antibodies:

• Sample Preparation:

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

  • Use fresh tissue when possible or flash-freeze and store at -80°C

  • Include positive and negative control samples

• Antibody Dilution:

  • Start with a 1:1000 dilution and optimize as needed

  • Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) to determine optimal concentration

• Blocking and Incubation:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Incubate with primary antibody overnight at 4°C

  • Wash thoroughly with TBST (at least 3 × 10 minutes)

  • Incubate with an appropriate secondary antibody (anti-rabbit IgG)

• Detection System:

  • For enhanced sensitivity, consider using chemiluminescent detection systems

  • Expose membranes for different durations to optimize signal-to-noise ratio

For validation, incorporate controls similar to those used in antibody binding studies with other targets, such as conducting an eleven-point 1:2 dilution series starting at 200 nM and measuring binding kinetics .

What approaches can I use for immunofluorescence localization with At2g29860 Antibody?

For subcellular localization studies of At2g29860 protein using immunofluorescence:

• Sample Fixation:

  • Fix Arabidopsis tissues or cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Consider comparing different fixation methods to optimize antigen preservation

• Antibody Application:

  • Apply At2g29860 Antibody at 1:100 to 1:500 dilution (optimize empirically)

  • Use fluorescently labeled secondary antibodies (e.g., Alexa Fluor 488 or 594)

  • Include co-staining with organelle markers (e.g., LAMP1 for lysosomes)

• Advanced Dual-Labeling:

  • For co-localization studies, use antibodies from different host species

  • Consider using Alexa Fluor-labeled primary antibodies for direct detection

  • Apply methodologies similar to those validated in dual-label internalization assays

• Controls and Validation:

  • Include negative controls (secondary antibody only)

  • Use blocking peptide controls to verify specificity

  • Compare staining patterns with related proteins or known interaction partners

How can I quantitatively assess At2g29860 protein expression levels?

For quantitative assessment of At2g29860 protein expression:

• Western Blot Quantification:

  • Use recombinant At2g29860 protein to generate a standard curve

  • Include housekeeping proteins (e.g., actin) as loading controls

  • Apply densitometry analysis using software such as ImageJ or ImageQuantTL

• ELISA Approaches:

  • Develop a sandwich ELISA using At2g29860 Antibody

  • Create a standard curve with recombinant protein

  • Calculate protein concentration in unknown samples by interpolation

• Flow Cytometry:

  • For single-cell analysis, use protocols similar to cell-based antibody binding studies

  • Consider an approach using an eleven-point 1:2 dilution series of antibody

  • Calculate median fluorescence intensities (MFIs) and derive apparent Kd values

How can I use At2g29860 Antibody in protein trafficking and internalization studies?

For studying protein trafficking and internalization:

• Antibody Internalization Assays:

  • Label At2g29860 Antibody with Alexa Fluor 488 or 594 using established conjugation protocols

  • Assess internalization using quenching approaches similar to those described for other antibodies

  • Consider developing internalization assays based on validated anti-Alexa Fluor mAbs that effectively quench cell surface-bound fluorescence

• Time-Course Analysis:

  • Monitor protein trafficking over time using live-cell imaging

  • Quantify internalization rates by measuring fluorescence intensity changes

  • Compare results with known regulatory pathways in Arabidopsis

• Co-Localization Studies:

  • Combine At2g29860 Antibody with markers for different cellular compartments

  • Use dual-labeling approaches to track protein movement

  • Apply methods similar to those used in validated proof-of-concept dual-label internalization assays

What approaches can I use to study At2g29860 in relation to hormone response pathways?

For investigating At2g29860 in the context of hormone response pathways:

• Expression Analysis in Hormone-Treated Samples:

  • Treat Arabidopsis with hormones such as indole-3-acetic acid (IAA) or brassinolide (BL)

  • Compare At2g29860 protein levels before and after treatment

  • Consider including known hormone-responsive genes/proteins as positive controls

• Co-Immunoprecipitation Studies:

  • Use At2g29860 Antibody to pull down the protein and its interaction partners

  • Identify potential interactions with components of hormone signaling pathways

  • Validate interactions using reciprocal co-IP or other protein-protein interaction assays

• Comparative Expression Analysis:

  • Analyze At2g29860 expression in relation to known hormone-regulated genes

  • Consider methodologies similar to those used for comprehensive transcript profiling in response to plant hormones

  • Determine if At2g29860 falls into categories similar to genes regulated at early or late stages in hormone response

How can I troubleshoot potential cross-reactivity issues with At2g29860 Antibody?

Cross-reactivity can be a significant challenge when working with antibodies in plant research. To address this:

• Validation in Knockout/Knockdown Lines:

  • Test the antibody in At2g29860 knockout or knockdown lines

  • Compare signal intensity with wild-type samples

  • Residual signal in knockout lines may indicate cross-reactivity

• Peptide Competition Assays:

  • Pre-incubate the antibody with excess recombinant At2g29860 protein

  • Apply the mixture to your samples

  • A significant reduction in signal confirms specificity

• Western Blot Analysis:

  • Look for additional bands that may indicate cross-reactivity

  • Compare observed molecular weight with predicted size

  • Consider testing in different tissues to identify tissue-specific cross-reactivity

• Specificity Testing:

  • Test against related proteins or homologs

  • Consider using approaches similar to kinetic measurements established with an Octet QK384 for other antibodies

  • Calculate binding parameters such as KD, kon, and koff to assess specificity

How should I analyze antibody binding kinetics for At2g29860 Antibody?

For rigorous analysis of At2g29860 Antibody binding kinetics:

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize recombinant At2g29860 protein onto biosensor surfaces

  • Measure association and dissociation rates using instruments like the Octet QK384

  • Apply methodologies similar to those used for anti-EphA2 mAbs with anti-human IgG Fc biosensors

• Data Analysis Parameters:

  • Conduct an eight-point two-fold titration series

  • Measure association for 5 minutes and dissociation for 20 minutes

  • Analyze and fit data globally using a 1:1 Langmuir binding model

  • Calculate KD by dividing kdis by kon

• Cell-Based Binding Studies:

  • Implement protocols similar to those described for other antibodies

  • Use a dilution series of Alexa Fluor-labeled At2g29860 Antibody

  • Measure median fluorescence intensities (MFIs) at each dilution

  • Plot data and derive apparent Kd using one-site specific binding with Hill slope equation

How can I interpret At2g29860 expression data in the context of plant developmental stages?

For meaningful interpretation of At2g29860 expression across developmental stages:

• Comparative Analysis:

  • Compare expression patterns with known developmental markers

  • Consider relationships to genes within functional categories such as cell wall modification, signaling, or stress response

  • Analyze potential co-expression with genes regulated at similar developmental stages

• Integration with Transcriptomic Data:

  • Correlate protein expression data with available transcriptome profiles

  • Consider methodologies similar to those used to identify genes up- or down-regulated at early and late stages in response to hormones

  • Develop integrated models of gene and protein expression dynamics

• Functional Classification:

  • Assign potential functions based on expression patterns

  • Consider classification systems similar to those used for hormone-regulated genes (e.g., categories A-M as in comprehensive hormone response studies)

  • Validate predictions through additional functional assays

What emerging technologies might enhance At2g29860 Antibody applications?

Several cutting-edge technologies could expand the utility of At2g29860 Antibody:

• Single-Cell Proteomics:

  • Apply At2g29860 Antibody in microfluidic single-cell Western blot systems

  • Develop methods for quantitative assessment of protein levels in individual cells

  • Integrate with single-cell transcriptomics for multi-omics approaches

• Super-Resolution Microscopy:

  • Optimize At2g29860 Antibody labeling for techniques like STORM, PALM, or STED

  • Achieve nanoscale resolution of protein localization

  • Combine with multi-color imaging for co-localization studies

• Proximity Labeling:

  • Develop At2g29860 Antibody conjugates with proximity labeling enzymes

  • Map protein interaction networks in living cells

  • Identify novel interaction partners and functional relationships

How might At2g29860 Antibody contribute to understanding plant stress responses?

The At2g29860 Antibody could provide valuable insights into plant stress responses through:

• Comparative Stress Studies:

  • Analyze expression and localization under different stress conditions

  • Compare with known stress-responsive proteins

  • Consider relationships to genes up- or down-regulated during stress responses

• Signaling Pathway Analysis:

  • Investigate potential involvement in hormone-mediated stress responses

  • Consider connections to genes regulated by hormones like IAA or BL

  • Develop models of At2g29860 function in stress signaling networks

• Transgenic Approaches:

  • Use At2g29860 Antibody to validate overexpression or knockdown phenotypes

  • Quantify protein levels in transgenic lines

  • Correlate protein expression with observed phenotypic changes