At4g08028 Antibody

What is the specificity of commercially available At4g08028 antibodies?

The specificity of At4g08028 antibodies is determined by thorough validation against both wild-type and knockout Arabidopsis lines. Current antibodies are developed using either synthetic peptides or recombinant protein approaches targeting specific epitopes of the At4g08028 protein. Bioinformatic analysis is employed to identify potential antigenic regions with less than 40% sequence similarity to other proteins to minimize cross-reactivity . When working with At4g08028 antibodies, researchers should verify specificity through Western blot testing against mutant backgrounds, as demonstrated with similar Arabidopsis proteins like AXR4, ACO2, and ARF19 .

What are the recommended applications for At4g08028 antibodies?

At4g08028 antibodies are suitable for several experimental techniques:

• Western blot (WB): Typically using dilutions of 1:5000-1:10000

• Immunofluorescence (IF): Recommended dilution around 1:200

• Chromatin Immunoprecipitation (ChIP): Typically using 2 μg per reaction

• Immunolocalization (IL): Similar to IF protocols with 1:200 dilution

For optimal results in Western blot applications, current protocols recommend using 20-80 μg of Arabidopsis total cell extract, separated on 6-10% SDS-PAGE, and blocking with 5% low-fat milk powder in TBS-TT (0.25% TWEEN20; 0.1% Triton-X) .

How should At4g08028 antibodies be stored for maximum stability?

Based on recommended practices for Arabidopsis antibodies:

After reconstitution, make aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality. Always spin tubes briefly before opening to collect material that might adhere to the cap or sides . For lyophilized antibodies, reconstitute in sterile water (typically 50 μl for 50 μg of antibody) .

How can I validate At4g08028 antibody specificity in my experimental system?

A comprehensive validation protocol includes:

• Western blot against mutant lines: Test antibody reactivity in At4g08028 knockout/knockdown lines

• Preabsorption test: Pre-incubate the antibody with excess purified antigen prior to use in experiments; a specific antibody will show reduced or eliminated signal

• Alternative antibody comparison: Compare results with antibodies targeting different epitopes of the same protein

• Cross-species reactivity: Test against homologous proteins in related plant species to evaluate specificity

For At4g08028, which is involved in mismatch repair pathways, validation in different tissues and developmental stages is critical since expression may vary significantly under different physiological conditions .

What controls should be included when using At4g08028 antibodies in immunoprecipitation experiments?

For robust immunoprecipitation results:

• Input control: 5-10% of the starting material

• IgG control: Non-specific antibody of the same isotype

• No-antibody control: Beads alone to assess non-specific binding

• Knockout/knockdown control: Material from plants lacking or having reduced At4g08028 expression

• Reciprocal IP: When studying interactions, confirm with antibodies against suspected interacting partners

In ChIP experiments with At4g08028 antibody, qPCR validation should be performed on mismatch repair-associated genomic regions, similar to validation approaches used for MSH4 ChIP-qPCR at crossover hotspot loci .

How should I design experiments to study At4g08028 localization patterns in different tissues?

For effective localization studies:

• Fixation optimization: For Arabidopsis tissues, use 4% paraformaldehyde fixation for 20-30 minutes at room temperature

• Tissue-specific protocols: Root tissues require different permeabilization conditions (0.1-0.3% Triton X-100) compared to leaf tissues

• Antibody concentration gradient: Test a range (1:100 to 1:500) to determine optimal signal-to-noise ratio

• Co-localization markers: Include established subcellular markers such as BiP (ER), γ-cop (Golgi), PM-ATPase (plasma membrane), or nuclear markers as appropriate

• Z-stack imaging: Collect multiple focal planes for comprehensive localization analysis

For quantitative analysis of At4g08028 expression, GUS reporter lines can be used to complement immunolocalization data, especially for examining expression changes under stress conditions .

What are the best approaches for studying At4g08028 protein interactions using antibody-based methods?

For protein interaction studies:

• Co-immunoprecipitation (Co-IP): Use At4g08028 antibody to pull down the protein complex, followed by Western blot analysis of suspected interacting partners

• Proximity Ligation Assay (PLA): Provides in situ detection of protein-protein interactions with spatial resolution

• Chromatin Immunoprecipitation (ChIP): For studying DNA-protein interactions relevant to At4g08028's role in mismatch repair

• Sequential ChIP: To identify co-occupancy of At4g08028 with other mismatch repair proteins at specific genomic loci

• Immunoprecipitation followed by mass spectrometry: For unbiased identification of interaction partners

When studying At4g08028 interactions, consider the relationship to cohesin subunits (like REC8) and nucleosome positioning, as observed with other mismatch repair proteins in Arabidopsis .

What are the common issues with At4g08028 antibody detection in Western blots and how can they be resolved?

For plant samples specifically, adding 2% PVPP (polyvinylpolypyrrolidone) to extraction buffers can help remove phenolic compounds that may interfere with antibody detection .

How can preexisting antibodies or interfering compounds be managed when using At4g08028 antibodies?

When dealing with preexisting antibodies or interfering compounds:

• Individual cutpoint determination: Set specific background thresholds for each sample based on pre-immune serum reactivity

• Competition assays: Develop assays that can detect specific anti-CDR antibodies in the presence of preexisting antibodies by using relevant control proteins

• Sample dilution optimization: Determine the minimal required dilution (MRD) that minimizes matrix effects while maintaining sensitivity

• Acid dissociation: For breaking up immune complexes that may mask epitopes, though this may not be appropriate if antibodies are acid-labile

• Trough level sampling: Collect samples at time points when interfering compounds are at their lowest concentration

How can At4g08028 antibodies be used to study chromatin associations during meiotic recombination?

For studying At4g08028's role in meiotic recombination:

• Synchronized meiocyte isolation: Optimize protocols to collect meiocytes at specific stages

• ChIP-seq analysis: Generate genome-wide binding profiles of At4g08028, similar to methodologies used for MSH4 ChIP-seq

• Sequential ChIP: Identify co-localization with other meiotic proteins

• Super-resolution microscopy: Use fluorescently labeled antibodies to visualize At4g08028 localization on meiotic chromosomes

• Quantitative analysis: Compare enrichment patterns between wild-type and mutant backgrounds

Analysis should focus on:

• Correlation with recombination hotspots

• Co-localization with cohesin complexes (REC8)

• Relationship to heterochromatin/euchromatin boundaries

• Association with DSB sites marked by SPO11-oligonucleotides

What experimental strategies can be employed to investigate At4g08028's role in DNA damage response using antibody-based approaches?

For DNA damage response studies:

• Immunofluorescence time course: Track At4g08028 localization after DNA damage induction (gamma irradiation, cisplatin, or MMC treatment)

• Proximity ligation assay (PLA): Detect interactions with known DNA damage response proteins

• ChIP-seq after DNA damage: Map changes in genome-wide binding profiles following genotoxic stress

• Phospho-specific antibodies: Develop antibodies that recognize post-translationally modified forms of At4g08028 after DNA damage

• FRAP (Fluorescence Recovery After Photobleaching): When combined with antibody visualization, can assess protein dynamics at damage sites

Compare findings with other mismatch repair proteins in plants, particularly examining changes in mobility or localization following exposure to genotoxic stress or during specific abiotic stress responses like salinity, which has been shown to affect MSH7 expression in Arabidopsis .

How can epitope-specific antibodies be designed to distinguish between At4g08028 and other closely related mismatch repair proteins?

For developing highly specific antibodies:

• Structure-based epitope selection: Use 3D structural information (when available) to identify exposed, unique regions

• Multiple sequence alignment: Compare At4g08028 with other MSH family members (MSH2, MSH4, MSH5, MSH7) to identify divergent regions

• Peptide array screening: Test multiple candidate epitopes in parallel

• Validation against knockout lines: Confirm specificity using genetic mutants for each MSH family member

• Cross-reactivity testing: Test against recombinant proteins of related MSH family members