The At5g44400 antibody is a polyclonal antibody targeting the protein product of the At5g44400 gene in Arabidopsis thaliana (Mouse-ear cress). This gene encodes BBE26, a member of the Berberine Bridge Enzyme (BBE)-like protein family, which is implicated in oxidative modifications of cell wall-derived oligosaccharides during plant immune responses .
Genomic Location: Chromosome 5, within a cluster of paralogous genes (At5g44360/BBE23, At5g44380/BBE24, At5g44390/BBE25, At5g44400/BBE26, At5g44410/BBE27) .
Functional Role: BBE26 is a flavin-dependent oxidase active on cellodextrins (CDs) and mixed-linked β-1→3/β-1→4-glucans (MLGs), generating oxidized oligosaccharides that modulate plant immune signaling .
Substrate Specificity:
BBE26 oxidizes cello-oligosaccharides (e.g., cellotriose, cellotetraose) and MLGs, producing aldonic acid-terminated glycans that reduce elicitor activity .
Enzymatic activity measured via HPAEC-PAD chromatography shows distinct oxidation profiles compared to related enzymes (e.g., CELLOX1 and CELLOX2) .
Role in Immunity:
Expression Analysis: Used to detect BBE26 protein levels in studies investigating plant responses to fungal pathogens (e.g., Botrytis cinerea) .
Localization Studies: Potential use in subcellular localization assays to determine tissue-specific expression patterns (e.g., root or leaf tissues) .
Specificity: The antibody is raised against a recombinant fragment of BBE26, ensuring minimal cross-reactivity with other BBE-like proteins .
Validation: Functional validation via enzymatic assays and gene silencing (e.g., CRISPR/Cas9 mutants) confirms antibody reliability in detecting BBE26 .
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.
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.
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 .
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:
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:
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
Based on transcriptomic analysis:
Expression correlation:
Pathway integration:
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
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
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:
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
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:
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
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
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
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
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