At5g02995 Antibody

What is the At5g02995 protein and why would researchers develop antibodies against it?

At5g02995 is an Arabidopsis thaliana gene encoding a galactose oxidase/kelch repeat protein . Researchers develop antibodies against this protein primarily for plant molecular biology investigations, particularly in studies examining:

• Protein expression patterns in different plant tissues

• Protein localization studies

• Protein-protein interaction analyses

• Functional characterization of kelch repeat proteins in plant development and stress responses

The antibody allows detection of the native protein in its cellular context, providing insights into its biological role that genomic analysis alone cannot reveal .

What validation methods should be employed before using the At5g02995 antibody?

Before using the At5g02995 antibody in critical experiments, researchers should perform multiple validation steps:

As indicated in the product specifications, this antibody has been validated for ELISA and Western blot applications, with specificity for Arabidopsis thaliana .

What are the optimal storage conditions for maintaining At5g02995 antibody activity?

To maintain optimal activity of the At5g02995 antibody:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles that can cause protein denaturation

• Store in the recommended buffer (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300)

• For short-term use (1-2 weeks), aliquot and store at 4°C

• For long-term stability, prepare small single-use aliquots before freezing

• Document the number of freeze-thaw cycles in laboratory records

Proper storage is critical for maintaining binding affinity and preventing non-specific background signal in experiments .

How should I determine the optimal working dilution for the At5g02995 antibody in Western blot applications?

Determining the optimal antibody concentration requires systematic titration:

• Prepare a dilution series of the antibody (e.g., 1:500, 1:1000, 1:2000, 1:5000, 1:10000)

• Use identical protein samples and blotting conditions for each dilution

• Process all blots simultaneously with identical detection conditions

• Evaluate signal-to-noise ratio across all dilutions

• Select the highest dilution that provides clear specific signal with minimal background

This approach is preferable to using a single manufacturer-recommended dilution as optimal concentrations can vary based on:

• Protein abundance in your specific samples

• Detection method sensitivity (chemiluminescence vs. fluorescence)

• Blocking reagents and incubation conditions used in your laboratory

What control samples are essential when using the At5g02995 antibody?

For rigorous experimental design with the At5g02995 antibody, include these controls:

These controls help distinguish genuine signals from artifacts and are essential for publication-quality data .

Can I use the At5g02995 antibody for co-immunoprecipitation experiments?

While the At5g02995 antibody is validated for ELISA and Western blot applications , its suitability for co-immunoprecipitation (Co-IP) requires empirical testing:

• The antibody's affinity (which determines its ability to pull down target protein) is a critical factor

• The epitope recognized must be accessible in the protein's native conformation

• Test precipitation efficiency by performing a pilot IP followed by Western blot

• Compare different lysis/binding buffers to optimize conditions

• Consider crosslinking the antibody to beads to prevent antibody contamination in the eluate

• Include appropriate negative controls (IgG control, lysate from knockout lines)

If the antibody successfully immunoprecipitates the target protein, proceed with co-IP experiments to identify interaction partners, followed by mass spectrometry or Western blot analysis .

How can I adapt the At5g02995 antibody for use in immunohistochemistry despite no formal validation for this application?

The At5g02995 antibody has not been formally validated for immunohistochemistry (IHC) , but researchers can systematically optimize conditions:

• Fixation optimization: Test multiple fixatives (4% paraformaldehyde, acetone, methanol) and fixation times

• Antigen retrieval methods: Compare heat-induced epitope retrieval (citrate buffer, pH 6.0) versus enzymatic retrieval

• Blocking optimization: Test different blockers (BSA, normal serum, commercial blocking reagents) to minimize background

• Antibody concentration gradient: Create a dilution series starting higher than Western blot concentration

• Signal amplification: Consider tyramide signal amplification if standard detection yields weak signals

• Critical controls: Include tissue from At5g02995 knockout plants as negative control

Document all optimization steps systematically. Successful adaptation enables valuable spatial expression pattern analysis that complements biochemical data .

What strategies can address epitope masking when At5g02995 forms protein complexes?

When At5g02995 interacts with other proteins, epitope masking can prevent antibody recognition, leading to false negative results. Consider these approaches:

• Denaturing conditions: For Western blots, ensure complete protein denaturation with SDS and reducing agents

• Native vs. denatured comparisons: Compare detection in native PAGE versus SDS-PAGE to identify masking

• Epitope mapping: Determine which region(s) of At5g02995 are recognized by the antibody using truncated protein variants

• Alternative antibodies: If available, use antibodies targeting different epitopes on the same protein

• Protein complex dissociation: Modify extraction buffers with higher salt concentration or mild detergents

• Crosslinking studies: Use reversible crosslinkers to capture complexes before disruption for analysis

These approaches help distinguish between protein absence and detection failure due to complex formation .

How can quantitative fluorescence-based methods be optimized for At5g02995 antibody applications?

To develop quantitative fluorescence-based detection with the At5g02995 antibody:

• Fluorophore selection: Choose fluorophores with appropriate spectral properties that don't overlap with plant autofluorescence

• Signal-to-noise optimization:

  • Implement rigorous background subtraction methods

  • Use spectral unmixing to separate specific signal from autofluorescence

• Standard curve generation: Create standards using recombinant At5g02995 protein

• Dynamic range determination: Establish the linear range of detection through serial dilutions

• Multiple technical replicates: Include at least 3-4 replicates per sample

• Image acquisition standardization:

  • Maintain consistent exposure settings

  • Use identical gain and offset settings across all samples

  • Implement flat-field correction for microscopy applications

This approach enables reliable quantification of At5g02995 protein levels across different experimental conditions .

How should I interpret unexpected molecular weight bands when using the At5g02995 antibody?

When Western blots show unexpected bands, systematic analysis is required:

Always compare observed patterns with available literature and transcript data to distinguish between artifacts and biologically relevant signals .

What approaches can resolve non-specific binding issues with the At5g02995 antibody?

When experiencing high background or non-specific binding:

• Blocking optimization:

  • Test different blocking agents (5% milk, 5% BSA, commercial blockers)

  • Increase blocking time (overnight at 4°C)

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Antibody dilution adjustment:

  • Increase antibody dilution gradually (use 2-5× higher dilution)

  • Prepare antibody in fresh blocking buffer with 0.1% Tween-20

• Washing modifications:

  • Increase number and duration of washes

  • Use higher salt concentration in wash buffers (up to 500 mM NaCl)

  • Add 0.1% SDS to wash buffer for stubborn non-specific binding

• Pre-adsorption technique:

  • Incubate antibody with protein extract from knockout plants

  • Remove bound antibodies by centrifugation before using supernatant

• Secondary antibody considerations:

  • Test alternative secondary antibodies

  • Use highly cross-adsorbed secondary antibodies

These approaches systematically identify and eliminate sources of non-specific binding .

How can I determine if cross-reactivity with related proteins is affecting my results with the At5g02995 antibody?

Cross-reactivity with related proteins can complicate data interpretation. Address this through:

• Sequence homology analysis:

  • Identify Arabidopsis proteins with sequence similarity to At5g02995

  • Focus on regions containing the immunizing peptide sequence

• Control experiments:

  • Test antibody against protein extracts from plants overexpressing related proteins

  • Use genetic knockout lines of At5g02995 to identify non-specific signals

• Competition assays:

  • Perform peptide competition with immunizing peptide

  • Include peptides from homologous regions of related proteins

• Immunodepletion:

  • Pre-clear samples using recombinant At5g02995 protein

  • Compare binding patterns before and after depletion

• Orthogonal detection methods:

  • Compare results with tagged protein expression

  • Use mass spectrometry to identify proteins in immunoprecipitated samples

This systematic approach distinguishes specific from cross-reactive signals, essential for accurate data interpretation .

How can I use the At5g02995 antibody to study protein-protein interactions within the kelch repeat protein family?

To investigate protein-protein interactions involving At5g02995:

• Co-immunoprecipitation (Co-IP):

  • Use the At5g02995 antibody to pull down the protein complex

  • Analyze co-precipitated proteins by mass spectrometry or Western blot

  • Validate with reverse Co-IP using antibodies against identified partners

• Proximity labeling approaches:

  • Create fusion proteins of At5g02995 with BioID or APEX2

  • Use the antibody to confirm expression of the fusion protein

  • Compare biotinylated proteins between experimental and control samples

• FRET-FLIM analysis:

  • Use the antibody to validate expression of fluorescently tagged At5g02995

  • Measure FRET between At5g02995 and candidate interaction partners

• Split-reporter assays:

  • Validate expression of fusion proteins with the antibody

  • Correlate reporter signal with protein expression levels

These complementary approaches provide robust evidence for specific protein-protein interactions involving At5g02995 .

What strategies can integrate the At5g02995 antibody with advanced microscopy techniques for protein localization studies?

Combining the At5g02995 antibody with advanced microscopy enables detailed protein localization analysis:

• Super-resolution microscopy:

  • Use fluorophore-conjugated secondary antibodies compatible with STORM, PALM, or STED

  • Implement drift correction and calibration standards for quantitative measurements

  • Compare apparent distributions between conventional and super-resolution imaging

• Correlative light and electron microscopy (CLEM):

  • Optimize fixation conditions compatible with both immunofluorescence and electron microscopy

  • Use gold-conjugated secondary antibodies for TEM visualization

  • Align fluorescence and EM images using fiducial markers

• Live-cell imaging validation:

  • Compare antibody-based localization in fixed cells with fluorescent protein fusions in live cells

  • Use the antibody to validate expression levels of tagged proteins

• Multiplexed imaging:

  • Combine the At5g02995 antibody with markers for specific subcellular compartments

  • Implement spectral unmixing for accurate signal separation

  • Quantify colocalization using appropriate statistical measures

These approaches provide complementary data on protein localization at different resolution scales .

How can cryoEM techniques be applied to characterize the structural epitopes recognized by the At5g02995 antibody?

Structural characterization of antibody-antigen interactions using cryoEM involves:

• Sample preparation optimization:

  • Form complexes between purified At5g02995 protein and antibody Fab fragments

  • Screen buffer conditions to ensure complex stability and homogeneity

  • Optimize vitrification parameters for ice thickness and particle distribution

• Data collection strategy:

  • Collect tilt series to address preferred orientation issues

  • Implement motion correction and CTF estimation protocols

  • Use appropriate electron dose to minimize radiation damage

• Image processing workflow:

  • Perform reference-free 2D classification to identify homogeneous particle populations

  • Generate ab initio 3D reconstructions

  • Apply focused refinement on the antibody-epitope interface

• Validation approaches:

  • Perform mutagenesis of predicted epitope residues to confirm structural model

  • Cross-validate with hydrogen-deuterium exchange mass spectrometry

  • Compare with computational docking predictions

This approach reveals the structural basis of antibody specificity and can guide the development of improved antibodies with enhanced properties .