At5g23035 Antibody

What is At5g23035 and why would researchers need antibodies against this protein?

At5g23035 encodes a putative membrane lipoprotein in Arabidopsis thaliana (mouse-ear cress), a widely used model organism in plant biology . Researchers require antibodies against this protein to:

• Study protein localization within cellular compartments

• Determine protein expression levels in different tissues or under various conditions

• Investigate protein-protein interactions

• Characterize protein function in biological pathways

Methodologically, when working with At5g23035 antibodies, researchers should first confirm the protein's expression pattern using techniques like RNA-seq or RT-PCR before proceeding with antibody-based detection methods to establish baseline expectations for protein abundance and distribution.

How should researchers validate an At5g23035 antibody before experimental use?

Antibody validation is crucial for ensuring experimental reproducibility. For At5g23035 antibody validation, researchers should follow these methodological steps:

• Western blot analysis using known positive controls: Use tissues known to express At5g23035, comparing band sizes to predicted molecular weights .

• Negative controls: Test the antibody against samples from knockout or knockdown lines where At5g23035 is not expressed .

• Absorption controls: Pre-incubate the antibody with purified At5g23035 protein or peptide antigen to confirm specificity .

• Cross-reactivity assessment: Test against related proteins or tissues from other plant species to evaluate specificity .

The table below summarizes key controls for antibody validation:

IB: Immunoblot, IHC: Immunohistochemistry

What information should be recorded about At5g23035 antibodies in laboratory notebooks?

Proper documentation is essential for reproducibility. For At5g23035 antibodies, record:

• Antibody source: Company name, catalog number, or laboratory source if custom-made

• Host species and clonality: Specify whether monoclonal or polyclonal, and the host animal

• Immunogen details: Peptide sequence or full protein information used to generate the antibody

• Lot number: Critical for tracing batch-to-batch variations

• Dilutions used: Record specific dilutions for each application (Western blot, immunohistochemistry, etc.)

• Incubation conditions: Temperature, duration, and buffer compositions

• Validation experiments performed: Reference to specific validation tests completed

• Target protein concentrations: Amount of protein loaded in each experiment

For newly developed antibodies against At5g23035, additional information should include the sequence used for immunization, UniProt number of the full-length protein, host, and bleed number .

What are the recommended applications for At5g23035 antibodies in plant research?

At5g23035 antibodies can be employed in various experimental applications:

• Western blotting: For quantifying protein expression levels and confirming protein size

  • Recommended protein load: 5-25 μg total protein depending on abundance

  • Use total protein staining (e.g., Ponceau S) for loading control rather than housekeeping proteins

• Immunohistochemistry/Immunocytochemistry: For determining subcellular localization

  • Fixation considerations specific to plant tissues must be optimized

  • Background autofluorescence from plant cell walls requires careful controls

• Co-immunoprecipitation: For studying protein-protein interactions

  • Consider using crosslinking agents for transient interactions

• Chromatin immunoprecipitation: If At5g23035 has DNA-binding functions

  • Optimize crosslinking conditions for plant tissues

• ELISA: For quantitative detection in plant extracts

Each application requires specific optimization for plant tissues, particularly considering the cell wall and vacuole structures unique to plant cells.

How should researchers report At5g23035 antibody use in publications?

Proper reporting of antibody details is crucial for reproducibility. When publishing research using At5g23035 antibodies, include:

• Complete antibody information:

  • Commercial source and catalog number or reference to production method if custom-made

  • Clone designation for monoclonal antibodies

  • Host species and isotype

  • UniProt identifier for the target protein (At5g23035)

• Validation evidence:

  • Description of validation experiments performed

  • Provision of full, unedited blot images as supplementary data

  • Clear labeling of specific and non-specific bands

  • Details on controls used to demonstrate specificity

• Experimental conditions:

  • Dilutions used for each application

  • Incubation conditions (time, temperature, buffers)

  • Protein amounts loaded for Western blots

  • Exposure times for imaging

For newly developed antibodies, include detailed information about the antigen used for immunization and comprehensive validation data .

What are the best strategies for addressing cross-reactivity in At5g23035 antibody experiments?

Cross-reactivity is a significant concern for plant protein antibodies due to gene duplication and protein families with similar domains. For At5g23035 antibodies:

• Epitope mapping and analysis:

  • Identify the exact epitope recognized by the antibody

  • Compare this sequence against other Arabidopsis proteins using bioinformatic tools

  • Consider using domain-specific antibodies that target unique regions of At5g23035

• Absorption assays with related proteins:

  • Express and purify related proteins

  • Pre-incubate antibody with these proteins to identify cross-reactivity

  • Quantify the degree of cross-reactivity using titration experiments

• Testing in multiple genetic backgrounds:

  • Use At5g23035 overexpression lines to confirm signal increase

  • Test in knockout/knockdown lines to confirm signal reduction

  • Compare with related gene knockouts to assess specificity

• Orthogonal validation approaches:

  • Use epitope-tagged versions of At5g23035 and compare localization/expression patterns

  • Employ mass spectrometry to confirm antibody-precipitated proteins

  • Use RNA-protein correlation studies to validate expression patterns

These approaches should be implemented systematically, with results documented to support antibody specificity claims.

How does protein glycosylation impact At5g23035 antibody recognition and what methods can address these challenges?

Plant proteins often undergo post-translational modifications, including glycosylation, which can affect antibody binding. For At5g23035:

• Effects of glycosylation on antibody recognition:

  • Glycosylation can mask epitopes, reducing antibody accessibility

  • Plant-specific glycosylation patterns may differ from the immunization host

  • Glycosylation can alter protein migration in SDS-PAGE

• Assessment methodologies:

  • Compare antibody reactivity on glycosylated versus deglycosylated samples using PNGase F treatment

  • Test antibody recognition using glycosylation pathway mutants

  • Generate antibodies against both glycosylated and non-glycosylated forms of the protein

• Experimental approaches:

  • For Western blotting: Deglycosylate samples before SDS-PAGE

  • For immunoprecipitation: Consider using denaturing conditions to expose hidden epitopes

  • For immunohistochemistry: Use antigen retrieval methods optimized for plant tissues

Research indicates that testing antibody reactivity with both glycosylated and deglycosylated protein extracts can provide valuable information about epitope accessibility and specificity .

What are the considerations for adapting At5g23035 antibodies for different experimental techniques?

Different experimental techniques require specific optimization for At5g23035 antibodies:

• Western blotting versus immunohistochemistry:

  • Western blotting involves denatured proteins while IHC uses native conformations

  • Epitopes accessible in one technique may be hidden in another

  • Test antibody functionality in both applications separately

• Fixation and sample preparation considerations:

  • For immunohistochemistry: Different fixatives (paraformaldehyde, glutaraldehyde) can affect epitope exposure

  • For electron microscopy: More stringent fixation may further alter epitope accessibility

  • For flow cytometry: Cell wall digestion protocols affect antibody penetration

• Buffer and blocking optimizations:

  • Plant-specific components in blocking buffers may be needed

  • Test different detergents and their concentrations for optimal signal-to-noise ratio

  • Consider plant-specific autofluorescence quenching for fluorescent applications

• Antigen retrieval methods for plant tissues:

  • Heat-induced epitope retrieval may need optimization for plant cell walls

  • Enzymatic treatments might be necessary for some applications

  • pH conditions should be systematically tested

Each application requires specific validation to ensure that the antibody recognizes At5g23035 appropriately under those particular experimental conditions .

What are the advantages of developing recombinant antibodies for At5g23035 compared to conventional antibodies?

Recombinant antibodies offer several advantages for At5g23035 research:

• Enhanced reproducibility:

  • Absolutely defined by amino acid sequence, ensuring batch-to-batch consistency

  • Digitally archived via DNA sequencing for unambiguous identification

  • Eliminates variation inherent in polyclonal antibody production

• Engineering possibilities:

  • Format flexibility: conversion between different species, isotypes, and formats

  • Creation of fusion proteins with reporter or affinity tags

  • Development of bispecific antibodies for complex experimental designs

• Production advantages:

  • Expression in chemically defined, serum-free mammalian systems

  • Lower endotoxin levels for in vivo applications

  • Sustainable, renewable source without animal immunization

• Experimental applications unique to recombinant antibodies:

  • Intracellular expression (intrabodies) for functional studies

  • Site-directed mutagenesis to improve specificity or affinity

  • Development of antibody libraries for epitope mapping

For plant proteins like At5g23035, recombinant antibodies can be engineered to recognize specific epitopes, reducing cross-reactivity with related plant proteins that often challenge conventional antibody approaches .

How can researchers incorporate computational prediction models to improve At5g23035 antibody design and characterization?

Computational approaches are increasingly valuable for antibody research against targets like At5g23035:

• Epitope prediction and antibody design:

  • Structure-based epitope prediction can identify accessible regions of At5g23035

  • Machine learning models can predict antibody binding affinity and specificity

  • Log-likelihood scores from generative models correlate with experimentally measured binding affinities

• Cross-reactivity assessment:

  • Computational screening against proteome databases identifies potential cross-reactive proteins

  • Structural modeling of antibody-antigen interactions predicts binding stability

  • Sequence alignment tools identify conserved domains requiring careful antibody design

• Integration of experimental and computational methods:

  • Machine learning models trained on experimental data improve prediction accuracy

  • Diffusion-based and graph-based modeling approaches provide complementary insights

  • Structure-based models generally outperform sequence-based ones for antibody design

Recent research demonstrates that computational techniques like DiffAbXL (a diffusion-based model) can generate antibody sequences with customized specificity profiles, potentially allowing for the development of highly specific antibodies against plant proteins like At5g23035 .