At5g44400 Antibody

What is the target protein of the At5g44400 Antibody?

The At5g44400 Antibody targets BERBERINE BRIDGE ENZYME-LIKE 26 (ATBBE26), a protein in Arabidopsis thaliana (Mouse-ear cress). This protein belongs to the berberine bridge enzyme-like family and is associated with the UniProt accession number Q9FKU8. The protein plays roles in plant development and has been identified in transcriptomic analyses related to flowering pathways . When selecting an antibody for this target, researchers should verify the specific epitope recognition to ensure detection of the protein of interest.

What expression patterns have been observed for At5g44400 in Arabidopsis?

Expression analyses have shown that At5g44400 (ATBBE26) is differentially regulated in flowering pathways. Microarray experiments comparing wild-type Col-0, ft-10 tsf-1, and pGAS1:FT ft-10 tsf-1 plants revealed that At5g44400 shows downregulation (logFC of -1.31) in the context of FLOWERING LOCUS T (FT) signaling . This suggests that ATBBE26 expression is potentially repressed during the floral transition. Researchers investigating this gene should consider temporal and tissue-specific expression patterns when designing experiments using the corresponding antibody.

What are appropriate positive and negative controls when using At5g44400 Antibody?

When working with At5g44400 Antibody, researchers should include:

Positive controls:

• Wild-type Arabidopsis tissue samples known to express ATBBE26

• Recombinant ATBBE26 protein (if available)

• Overexpression lines of At5g44400

Negative controls:

• At5g44400 knockout or knockdown lines

• Pre-immune serum controls

• Secondary antibody-only controls

• Blocking peptide competition assays to confirm specificity

These controls help validate antibody specificity and experimental conditions, particularly important when interpreting complex expression patterns in plant developmental studies .

How should western blot protocols be optimized for At5g44400 detection in Arabidopsis tissues?

For optimal western blot detection of ATBBE26 (At5g44400) in Arabidopsis tissues:

• Sample preparation:

  • Extract proteins from relevant tissues (leaves or shoot apices are recommended based on expression data)

  • Use extraction buffers containing protease inhibitors to prevent degradation

  • Consider tissue-specific optimization as At5g44400 shows differential expression patterns

• Electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Employ wet transfer methods for higher molecular weight proteins

  • Consider transfer time adjustments based on protein size (approximately 45-60 kDa expected)

• Antibody incubation:

  • Test multiple dilutions (1:500 to 1:5000) to determine optimal concentration

  • Extend primary antibody incubation time (overnight at 4°C) for improved sensitivity

  • Use 5% BSA or milk for blocking to minimize background

• Detection strategies:

  • Compare chemiluminescence vs. fluorescence-based detection methods

  • Consider the expected ELISA titer of 10,000 (similar to other plant antibodies) when optimizing detection parameters

What considerations should be made when using At5g44400 Antibody for immunohistochemistry?

When performing immunohistochemistry with At5g44400 Antibody:

• Fixation optimization:

  • Test both cross-linking (paraformaldehyde) and precipitating (acetone) fixatives

  • Consider that the GUS staining protocol using 90% acetone fixation for 30 minutes on ice has proven effective for other plant proteins

  • Optimize fixation time to preserve epitope accessibility while maintaining tissue structure

• Antigen retrieval:

  • Evaluate the necessity of antigen retrieval methods for improved signal

  • Consider citrate buffer (pH 6.0) heat-induced epitope retrieval if initial results show weak signals

  • Test enzymatic retrieval methods if heat-induced methods prove unsuccessful

• Detection systems:

  • Compare DAB vs. fluorescence-based detection systems

  • For fluorescence, consider autofluorescence controls crucial for plant tissues

  • Use appropriate mounting media to preserve signal over time

• Tissue-specific considerations:

  • Based on microarray data showing differential expression in flowering-related tissues, focus on leaf veins and shoot apical meristems

  • Consider developmental stage carefully, as At5g44400 expression changes during flowering transitions

How can researchers validate the specificity of At5g44400 Antibody in their experimental system?

To validate At5g44400 Antibody specificity:

• Genetic approaches:

  • Compare antibody signal in wild-type vs. knockout/knockdown lines

  • Test in overexpression lines to confirm signal enhancement

  • Consider CRISPR-Cas9 edited lines with epitope modifications

• Biochemical validation:

  • Perform peptide competition assays using the immunizing peptides

  • Conduct immunoprecipitation followed by mass spectrometry

  • Compare results across multiple antibody preparations targeting different epitopes, similar to the approach used for other plant proteins with N-terminal, C-terminal, and mid-region targeting antibodies

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins of related BBE family members

  • Evaluate potential cross-reactivity with the related proteins in the berberine bridge enzyme family

  • Consider sequence alignment analysis to predict potential cross-reactivity

• Technical validation:

  • Compare antibody performance across multiple lots

  • Validate across different experimental techniques (western blot, immunoprecipitation, immunohistochemistry)

  • Document batch-to-batch variation for reproducible research

How does At5g44400 (ATBBE26) relate to flowering pathways in Arabidopsis?

Based on transcriptomic analysis:

• Expression correlation:

  • At5g44400 shows negative correlation with FLOWERING LOCUS T (FT) expression (logFC of -1.31 in comparative microarray experiments)

  • This suggests potential roles in flowering time regulation or floral development

• Pathway integration:

  • The downregulation of At5g44400 occurs alongside upregulation of key flowering genes including FT (logFC 6.03), FUL (logFC 2.83), and SEP3 (logFC 2.66)

  • This expression pattern suggests At5g44400 may act as a negative regulator in flowering pathways

• Experimental approaches:

  • Researchers should consider temporal expression analysis using At5g44400 Antibody during floral transition

  • Combine protein-level studies (using the antibody) with RT-qPCR analysis similar to methods described for SWEET genes

  • Design experiments that capture developmental time points before, during, and after floral transition

• Research applications:

  • The antibody can be used to track protein abundance changes during developmental transitions

  • Chromatin immunoprecipitation (ChIP) approaches may reveal regulatory interactions if ATBBE26 has DNA-binding properties

How can researchers interpret contradictory results when using At5g44400 Antibody?

When facing contradictory results:

• Technical considerations:

  • Verify antibody performance with appropriate controls and validation steps

  • Consider epitope masking due to protein interactions or post-translational modifications

  • Evaluate potential protein degradation during sample preparation

• Biological variables:

  • Assess developmental timing carefully, as At5g44400 expression varies during development

  • Consider environmental conditions, as plant proteins often show condition-dependent expression

  • Evaluate genetic background effects, particularly in mutant or transgenic lines

• Experimental approach comparison:

  • Compare protein detection (antibody-based) with transcript analysis (RT-qPCR)

  • Use multiple antibodies targeting different epitopes of the same protein when available

  • Implement complementary techniques like mass spectrometry to validate protein identity

• Systematic troubleshooting:

  • Document experimental conditions thoroughly to identify variables

  • Consider plant growth conditions, harvesting time, and tissue selection

  • Evaluate statistical approaches and sample size adequacy

What methodological approaches can be used to study At5g44400 protein-protein interactions?

For protein interaction studies:

• Co-immunoprecipitation (Co-IP):

  • Use At5g44400 Antibody to isolate native protein complexes

  • Optimize lysis buffers to preserve interactions (test multiple detergent concentrations)

  • Consider crosslinking approaches to capture transient interactions

  • Analyze precipitated complexes by mass spectrometry

• Proximity labeling approaches:

  • Generate fusion proteins (At5g44400-BioID or At5g44400-TurboID)

  • Use the antibody to confirm expression of fusion proteins

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

• Yeast two-hybrid validation:

  • Use antibody to validate expression of identified interactors in planta

  • Compare interaction networks across developmental stages

  • Focus on proteins involved in flowering pathways based on expression data

• In situ interaction analysis:

  • Implement proximity ligation assays using At5g44400 Antibody and antibodies against potential interactors

  • Perform co-localization studies in plant tissues

  • Consider BiFC (Bimolecular Fluorescence Complementation) validation of key interactions

What approaches can address non-specific binding when using At5g44400 Antibody?

To minimize non-specific binding:

• Blocking optimization:

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

  • Extend blocking time (1-3 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to washing buffers to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform dilution series to find optimal concentration

  • Consider that ELISA titers around 10,000 suggest dilutions of 1:1000-1:5000 may be appropriate

  • Pre-absorb antibody with plant extract from knockout lines if available

• Cross-reactivity mitigation:

  • Pre-incubate antibody with recombinant homologous proteins

  • Use high stringency washing conditions (increased salt concentration)

  • Consider affinity purification of polyclonal antibodies if cross-reactivity persists

• Technical adaptations:

  • For western blots, extend washing steps and increase detergent concentration

  • For immunohistochemistry, include appropriate permeabilization steps

  • Consider monoclonal antibody approaches for highly specific applications

How can researchers effectively use At5g44400 Antibody in chromatin immunoprecipitation studies?

For ChIP applications:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (1-3%) and crosslinking times

  • Consider dual crosslinking with DSG followed by formaldehyde for improved efficiency

  • Optimize quenching conditions to prevent over-crosslinking

• Chromatin preparation:

  • Evaluate sonication conditions specifically for plant tissues

  • Verify chromatin fragment size (200-500 bp optimal for most applications)

  • Optimize nuclear isolation protocols for plant tissues

• Immunoprecipitation considerations:

  • Test different antibody amounts (2-10 μg per reaction)

  • Evaluate various protein A/G beads and blocking conditions

  • Include appropriate controls (IgG, input, no antibody)

• Data analysis approaches:

  • Design primers for potential binding regions based on known BBE family binding motifs

  • Consider genome-wide approaches (ChIP-seq) for unbiased binding site identification

  • Integrate with RNA-seq data to correlate binding with gene expression changes

What quantitative approaches can measure At5g44400 protein abundance in different tissues?

For quantitative protein analysis:

• Quantitative western blotting:

  • Use recombinant ATBBE26 protein standards for absolute quantification

  • Implement internal loading controls appropriate for plant tissues

  • Consider fluorescence-based detection for wider dynamic range

  • Perform technical and biological replicates (minimum three biological replicates as used in related studies)

• Mass spectrometry approaches:

  • Develop selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) assays

  • Use stable isotope-labeled peptide standards for absolute quantification

  • Target unique peptides from ATBBE26 sequence for specificity

• ELISA development:

  • Establish sandwich ELISA using capture and detection antibodies

  • Develop competitive ELISA for higher sensitivity

  • Generate standard curves using recombinant protein

• Protein normalization considerations:

  • Account for tissue-specific differences in extraction efficiency

  • Consider developmental stage variations in total protein content

  • Normalize to consistent housekeeping proteins across samples

How can At5g44400 Antibody be used in single-cell protein analysis of plant tissues?

For single-cell applications:

• Immunofluorescence microscopy:

  • Optimize tissue clearing methods compatible with antibody epitope preservation

  • Implement optical sectioning techniques (confocal, light sheet microscopy)

  • Consider signal amplification methods for low abundance proteins

• Flow cytometry applications:

  • Develop protoplast isolation protocols optimized for protein preservation

  • Implement intracellular staining protocols for fixed protoplasts

  • Combine with cell type-specific markers for population analysis

• Spatial proteomics approaches:

  • Use the antibody in laser capture microdissection workflows

  • Implement imaging mass cytometry with metal-conjugated antibodies

  • Consider integration with single-cell transcriptomics data

• Technical considerations:

  • Validate antibody specificity at single-cell resolution

  • Develop appropriate controls for autofluorescence in plant tissues

  • Consider signal-to-noise optimization for rare cell populations

What considerations should be made when using At5g44400 Antibody across different Arabidopsis ecotypes?

For cross-ecotype studies:

• Sequence variation analysis:

  • Analyze At5g44400 sequence conservation across ecotypes

  • Identify potential epitope variations that might affect antibody binding

  • Consider using antibodies targeting highly conserved regions

• Expression pattern comparison:

  • Document baseline expression differences between ecotypes

  • Consider developmental timing variations between ecotypes

  • Normalize data appropriately when making cross-ecotype comparisons

• Experimental design adaptations:

  • Include ecotype-specific controls in each experiment

  • Consider using multiple antibodies targeting different epitopes

  • Validate findings with complementary methods (RT-qPCR, RNA-seq)

• Data interpretation considerations:

  • Account for natural variation in protein abundance

  • Consider post-translational modification differences between ecotypes

  • Document ecotype-specific protein interaction networks