BGAL17 Antibody

What is BGAL17 and what biological functions does it serve in Arabidopsis thaliana?

BGAL17 is a member of the beta-galactosidase family in Arabidopsis thaliana (Mouse-ear cress). Beta-galactosidases are enzymes that catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. In plants, these enzymes are involved in cell wall metabolism, particularly in the modification of cell wall polysaccharides during growth, development, and stress responses. The BGAL17 protein likely contributes to cell wall remodeling processes, similar to other members of this family such as BXL1 and BXL4, which have been shown to possess both xylosidase and arabinosidase activities . Though the specific role of BGAL17 has not been as thoroughly characterized as some other family members, its function can be inferred from related proteins in the same family that participate in cell wall structure modifications during plant development and stress responses.

How does the BGAL17 Antibody differ from other beta-galactosidase antibodies used in plant research?

The BGAL17 Antibody is specifically raised against the recombinant Arabidopsis thaliana BGAL17 protein and is a rabbit polyclonal antibody . Unlike antibodies targeting other beta-galactosidases, this antibody has been developed to recognize epitopes specific to BGAL17, allowing researchers to distinguish it from other members of the beta-galactosidase family. This specificity is crucial when investigating the unique functions of BGAL17 compared to related proteins like BXL1-BXL7.

The antibody is antigen-affinity purified, which enhances its specificity and reduces cross-reactivity with other proteins . While other beta-galactosidase antibodies may target conserved domains across the family, the BGAL17 Antibody enables targeted studies of this specific protein's expression patterns, localization, and functional roles in various plant tissues and under different experimental conditions.

What are the validated applications for BGAL17 Antibody in plant molecular biology research?

The BGAL17 Antibody has been validated for several research applications, primarily ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blotting (WB) for antigen identification . These validated applications provide researchers with reliable tools for:

• Quantitative measurement of BGAL17 protein levels in plant tissue extracts through ELISA

• Detection of BGAL17 protein in complex samples and determination of its molecular weight through Western Blotting

• Monitoring changes in BGAL17 expression during different developmental stages or in response to stress conditions

While not explicitly stated in the search results, similar antibodies in this class may also be compatible with immunohistochemistry (IHC) for localization studies and immunoprecipitation (IP) for protein-protein interaction analyses, though researchers should perform validation studies before applying the antibody to these purposes.

How can BGAL17 Antibody be effectively used to investigate cell wall remodeling during pathogen response in Arabidopsis?

The BGAL17 Antibody can be instrumental in investigating cell wall remodeling during pathogen response by enabling researchers to track changes in BGAL17 protein levels and localization. Drawing from studies with related proteins like BXL4, which is involved in immunity against pathogens like Botrytis cinerea , a comprehensive research approach would include:

• Temporal expression analysis: Using Western blotting with the BGAL17 Antibody to monitor changes in protein expression at different time points following pathogen challenge.

• Subcellular localization studies: Combining the antibody with cellular fractionation techniques to determine if BGAL17 localizes to the apoplast during infection, similar to BXL4 .

• Comparative analysis with known defense-related beta-galactosidases: Simultaneous detection of BGAL17 and other family members like BXL4 to identify potential functional redundancy or specialization in pathogen response.

• Co-localization with cell wall modifications: Using the antibody in conjunction with cell wall polysaccharide-specific probes to determine if BGAL17 associates with specific cell wall components during pathogen attack.

The BXL4 protein has been shown to be induced upon infection with necrotrophic fungal pathogens in a jasmonoyl isoleucine-dependent manner . Researchers could investigate whether BGAL17 follows similar expression patterns and regulatory mechanisms during pathogen infection.

What are the most effective experimental designs for studying BGAL17's potential role in plant immunity?

Based on successful approaches used with related proteins like BXL4 , the following experimental design would be effective for studying BGAL17's role in plant immunity:

Table 1: Comprehensive Experimental Design for BGAL17 Immunity Studies

For pathogen challenge experiments, researchers should conduct both drop inoculation assays (measuring lesion diameter) and spray inoculation followed by qPCR quantification of pathogen biomass, as these complementary approaches have proven effective in studies of BXL4 . Additionally, analyzing the expression of defense marker genes like JAZ10, PDF1.2, and PAD3 in wild-type and bgal17 mutant plants would reveal connections to established immune pathways.

How can researchers differentiate between overlapping functions of BGAL17 and other beta-galactosidases/xylosidases in cell wall metabolism?

Differentiating between overlapping functions of BGAL17 and related enzymes requires a multi-faceted approach utilizing the BGAL17 Antibody alongside genetic and biochemical techniques:

• Genetic redundancy analysis: Generate and characterize single, double, and higher-order mutants of BGAL17 with other family members (BXL1-BXL7). Phenotypic analysis of these mutant combinations would reveal functional overlap or specialization.

• Substrate specificity determination: Perform in vitro enzymatic assays with immunopurified BGAL17 (using the antibody) to define its substrate preference compared to other family members. BXL1 and BXL4 have been shown to possess both xylosidase and arabinosidase activities , and determining if BGAL17 has similar dual functionality would be valuable.

• Expression domain mapping: Use the BGAL17 Antibody for immunolocalization studies to determine if its tissue and subcellular distribution overlap with other family members. BXL4 localizes to the apoplast , and determining whether BGAL17 shares this localization would provide insights into potential functional redundancy.

• Conditional complementation tests: Express BGAL17 in mutants of other family members (e.g., bxl1, bxl4) under various promoters and assess phenotypic rescue. For example, BXL4 expression rescued the bxl1 mutant phenotype in seed coat epidermal cells , suggesting functional similarity despite different expression patterns.

• Protein interaction partners: Use the BGAL17 Antibody for co-immunoprecipitation experiments to identify unique and shared interaction partners with other family members, potentially revealing distinct biological contexts for seemingly similar enzymatic activities.

What are the optimal sample preparation protocols for detecting BGAL17 in Arabidopsis tissues using Western blotting?

For optimal detection of BGAL17 in Arabidopsis tissues using Western blotting, researchers should follow this comprehensive protocol:

Extraction Buffer Composition:

• 50 mM Tris-HCl (pH 7.5)

• 150 mM NaCl

• 1% Triton X-100

• 0.5% sodium deoxycholate

• 0.1% SDS

• 1 mM EDTA

• Protease inhibitor cocktail

• 1 mM PMSF (added fresh)

• 5 mM DTT (added fresh)

Sample Preparation Procedure:

• Harvest plant tissue (100-200 mg) and immediately flash-freeze in liquid nitrogen.

• Grind tissue to fine powder using mortar and pestle under liquid nitrogen.

• Add 400-500 μl of extraction buffer per 100 mg tissue.

• Homogenize thoroughly and incubate on ice for 30 minutes with occasional vortexing.

• Centrifuge at 13,000 × g for 20 minutes at 4°C.

• Transfer supernatant to a fresh tube and quantify protein concentration.

• Mix protein samples with 4× Laemmli buffer and heat at 70°C for 10 minutes (avoid boiling as it may cause aggregation of membrane-associated glycosidases).

• Load 20-50 μg of protein per lane on an 8-10% SDS-PAGE gel.

Western Blotting Parameters:

• Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer containing 20% methanol.

• Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Incubate with BGAL17 Antibody at 1:1000 dilution in blocking buffer overnight at 4°C.

• Wash 4× with TBST, 5 minutes each.

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature.

• Wash 4× with TBST, 5 minutes each.

• Develop using enhanced chemiluminescence reagents.

This protocol accounts for the potential membrane association of BGAL17 and preserves its epitopes for optimal antibody recognition, based on successful approaches used with related plant glycosidases like BXL4 .

How should researchers optimize immunohistochemistry protocols to visualize BGAL17 localization in plant tissues?

While the BGAL17 Antibody has been primarily validated for ELISA and Western blotting , researchers can adapt the following optimized immunohistochemistry protocol to visualize BGAL17 localization in plant tissues:

Tissue Fixation and Embedding:

• Fix freshly harvested tissue in 4% paraformaldehyde in PBS (pH 7.4) under vacuum for 1 hour, then overnight at 4°C.

• Wash tissues 3× in PBS, 10 minutes each.

• Dehydrate through an ethanol series (30%, 50%, 70%, 90%, 95%, 100%, 100%), 1 hour each.

• Clear with histoclear:ethanol series (25:75, 50:50, 75:25, 100:0), 1 hour each.

• Infiltrate with paraffin:histoclear mixtures (25:75, 50:50, 75:25), 2 hours each, followed by 100% paraffin overnight.

• Embed tissues in fresh paraffin and section at 8-10 μm thickness.

Immunohistochemistry Procedure:

• Deparaffinize sections with histoclear (2× for 10 minutes) and rehydrate through a descending ethanol series.

• Perform antigen retrieval by heating sections in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10 minutes.

• Cool slides to room temperature and wash 3× in PBS.

• Block with 5% BSA, 0.3% Triton X-100 in PBS for 1 hour at room temperature.

• Incubate with BGAL17 Antibody (1:100 to 1:500 dilution range) in blocking solution overnight at 4°C.

• Wash 4× in PBS with 0.1% Tween-20.

• Incubate with fluorophore-conjugated anti-rabbit secondary antibody (1:200) for 2 hours at room temperature.

• Wash 4× in PBS with 0.1% Tween-20.

• Counterstain with DAPI (1 μg/ml) for 10 minutes.

• Mount with anti-fade mounting medium.

Validation Controls:

• Negative control: Primary antibody omission

• Absorption control: Pre-incubate BGAL17 Antibody with excess antigen

• Positive control: Tissues with confirmed BGAL17 expression

• Comparative control: Parallel staining with antibodies against known cell wall proteins

This protocol incorporates elements that have been successful for localizing related cell wall enzymes like BXL4, which was found to localize to the apoplast .

What considerations should be taken when using BGAL17 Antibody for co-immunoprecipitation studies?

When using BGAL17 Antibody for co-immunoprecipitation (co-IP) studies to identify interaction partners, researchers should consider these critical factors:

Pre-IP Considerations:

• Antibody validation: Confirm the specificity of the BGAL17 Antibody via Western blot before attempting co-IP.

• Crosslinking assessment: Determine whether chemical crosslinking (e.g., formaldehyde, DSP) is necessary to capture transient interactions.

• Extraction conditions: Test multiple extraction buffers with varying detergent compositions, as BGAL17 is likely associated with membranes or cell walls.

Optimized Co-IP Protocol:

Extraction Buffer Options:

• Mild buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, protease inhibitors

• Intermediate buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitors

• Stringent buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, protease inhibitors

Procedure:

• Extract proteins from 1-2 g plant tissue using selected buffer (10 ml per g).

• Clear lysate by centrifugation (13,000 × g, 20 minutes, 4°C).

• Pre-clear with Protein A/G beads for 1 hour at 4°C.

• Incubate 1 mg protein with 5-10 μg BGAL17 Antibody overnight at 4°C with gentle rotation.

• Add 50 μl Protein A/G beads and incubate for 3 hours at 4°C.

• Wash beads 5× with extraction buffer.

• Elute bound proteins with 0.1 M glycine (pH 2.5) or by boiling in SDS sample buffer.

• Analyze by SDS-PAGE followed by silver staining and mass spectrometry.

Critical Quality Controls:

• IgG isotype control to identify non-specific binding

• Input sample (5-10%) to confirm target protein presence

• Supernatant after IP to assess depletion efficiency

• Reciprocal co-IP with antibodies against identified interaction partners

• Validation in knockout/knockdown lines

Given the potential function of BGAL17, researchers should particularly focus on detecting interactions with other cell wall-modifying enzymes and structural components, as seen in studies of related proteins .

What are common issues encountered when working with BGAL17 Antibody and how can they be addressed?

Researchers working with BGAL17 Antibody may encounter several technical challenges. Here are common issues and their solutions:

Table 2: Troubleshooting Guide for BGAL17 Antibody Applications

Special considerations for BGAL17:

• As a potential cell wall-associated protein, BGAL17 may require enhanced extraction methods. Use of cell wall degrading enzymes (e.g., cellulase, pectinase) in a sequential extraction protocol may improve yields.

• The glycosylation status of BGAL17 might affect antibody recognition. Treatment with deglycosylation enzymes prior to immunodetection can help determine if glycosylation is affecting antibody binding.

• Based on studies with related proteins like BXL4, consider that BGAL17 might be secreted to the apoplast , requiring specific extraction approaches for extracellular proteins.

How can researchers effectively analyze BGAL17 expression changes during stress responses and developmental transitions?

To effectively analyze BGAL17 expression changes during stress responses and developmental transitions, researchers should implement a multi-level analysis approach:

Comprehensive Expression Analysis Strategy:

• Transcript-level analysis:

  • RT-qPCR using gene-specific primers for BGAL17

  • RNA-seq analysis with specific focus on BGAL17 and related family members

  • Comparison with known stress-responsive genes (like the demonstrated upregulation of BXL4 after Botrytis cinerea infection and wounding )

• Protein-level analysis:

  • Quantitative Western blotting using BGAL17 Antibody with appropriate loading controls

  • ELISA-based quantification for higher throughput analysis

  • Consideration of tissue-specific extraction protocols to account for potential compartmentalization

• Activity-based analysis:

  • Enzymatic assays using specific substrates to measure β-galactosidase activity

  • In-gel activity assays to distinguish BGAL17 activity from other family members

  • Correlation of enzyme activity with protein levels detected by the antibody

Data Normalization and Interpretation:

For meaningful comparisons across experimental conditions, normalize BGAL17 levels to:

• Constitutively expressed reference genes/proteins for each specific condition

• Total protein content using methods like Bradford assay

• Activity of non-stress responsive enzymes as internal controls

Statistical Analysis Framework:

• Perform time-course experiments with at least 3-5 biological replicates

• Apply appropriate statistical tests (ANOVA with post-hoc tests for multiple comparisons)

• Use principal component analysis to identify patterns in expression across multiple stress conditions

• Consider machine learning approaches for identifying complex regulatory patterns

This comprehensive approach will allow researchers to distinguish BGAL17-specific responses from general stress responses and to identify potential unique roles compared to other family members like BXL4, which has demonstrated involvement in pathogen defense .

What advanced analytical techniques can be combined with BGAL17 Antibody to gain insights into its functional interactions?

Combining BGAL17 Antibody with advanced analytical techniques can provide deeper insights into its functional interactions and biological roles. Here are sophisticated approaches:

1. Proximity-dependent labeling techniques:

• BioID or TurboID: Fuse biotin ligase to BGAL17 to biotinylate proximal proteins in living cells

• APEX2 proximity labeling: Generate an APEX2-BGAL17 fusion to biotinylate neighboring proteins upon H₂O₂ addition

• Validation: Confirm interactions using BGAL17 Antibody in co-immunoprecipitation or co-localization studies

2. Advanced microscopy approaches:

• Super-resolution microscopy: Combine BGAL17 Antibody with techniques like STORM or PALM to visualize nanoscale localization

• FRET analysis: Use fluorophore-conjugated BGAL17 Antibody with antibodies against potential interaction partners

• Live-cell imaging: Track dynamic changes in BGAL17 localization during stress responses using fluorescently-tagged nanobodies derived from the BGAL17 Antibody

3. Multi-omics integration:

• Immunoprecipitation followed by mass spectrometry (IP-MS): Use BGAL17 Antibody to pull down protein complexes for proteomics analysis

• ChIP-seq equivalent for protein-polysaccharide interactions: Adapt chromatin immunoprecipitation approaches to study BGAL17 interaction with cell wall components

• Correlation of proteomics and metabolomics data: Link BGAL17 protein levels with changes in cell wall composition

4. Functional assays with spatial resolution:

• Cell wall fractionation followed by immunodetection: Determine which cell wall fractions contain BGAL17

• Enzyme activity assays on tissue sections: Couple BGAL17 Antibody staining with in situ enzyme activity detection

• Atomic force microscopy: Combine with immunogold labeling to correlate BGAL17 presence with nanomechanical properties of cell walls

5. System-level analysis:

• Interactome mapping: Place BGAL17 in the context of plant cell wall remodeling networks

• Co-expression network analysis: Identify genes consistently co-regulated with BGAL17

• Comparative analysis with related proteins: Context-specific comparison with BXL1-BXL7 to identify unique vs. redundant functions, particularly focusing on the well-characterized BXL4's role in plant immunity

Integrating these approaches with traditional antibody applications will provide a comprehensive understanding of BGAL17's functional roles in plant biology, potentially revealing previously unrecognized connections to stress responses and developmental processes.

What are the current methodological limitations in studying BGAL17 function, and how might they be overcome?

Current methodological limitations in studying BGAL17 function include:

1. Functional redundancy challenges:

• Limitation: Multiple beta-galactosidases with potentially overlapping functions complicate phenotypic analysis of single mutants.

• Solution: Generate higher-order mutants combining bgal17 with mutations in related genes. Use CRISPR/Cas9 to create multiplex mutants targeting several family members simultaneously.

2. Substrate specificity determination:

• Limitation: The exact substrates of BGAL17 in plant cell walls remain undefined.

• Solution: Develop activity-based protein profiling probes specific for beta-galactosidases. Combine with BGAL17 Antibody for immunoprecipitation followed by detailed enzymatic characterization using defined oligosaccharide substrates.

3. Temporal and spatial resolution:

• Limitation: Traditional methods lack the resolution to capture dynamic changes in BGAL17 localization and activity.

• Solution: Develop fluorescent activity-based probes that can track BGAL17 activity in living cells. Create conditional expression systems to manipulate BGAL17 levels in specific tissues.

4. In vivo relevance of biochemical findings:

• Limitation: In vitro enzymatic assays may not reflect actual activity in the complex cell wall environment.

• Solution: Develop cell wall models that better represent the in vivo substrate presentation. Use advanced microscopy techniques with the BGAL17 Antibody to visualize enzyme-substrate interactions in minimally disrupted cell walls.

5. Integration with cell wall mechanics:

• Limitation: Connecting biochemical activities to physical cell wall properties remains challenging.

• Solution: Combine BGAL17 functional studies with atomic force microscopy and other mechanical testing of cell walls in wild-type and mutant plants.

These approaches would build upon successful strategies used with related proteins like BXL4, which was shown to localize to the apoplast and contribute to immunity against B. cinerea through possible modification of cell wall polysaccharides .

How might BGAL17 be involved in novel plant immunity pathways based on findings from related glycosidases?

Based on findings from related glycosidases like BXL4, BGAL17 may participate in plant immunity through several mechanisms that warrant investigation:

• Cell wall integrity surveillance: Similar to BXL4's role in defense against Botrytis cinerea , BGAL17 might modify cell wall polysaccharides during pathogen attack, either restricting pathogen invasion or releasing oligosaccharide fragments that serve as damage-associated molecular patterns (DAMPs).

• Hormone-dependent defense regulation: BXL4 expression is induced after wounding in a jasmonate-dependent manner . Researchers should investigate whether BGAL17 is similarly regulated by defense hormones and contributes to hormone-mediated immunity.

• Systemic immunity signaling: BXL4 shows increased expression in distal leaves after local pathogen infection , suggesting a role in systemic resistance. BGAL17 might similarly contribute to long-distance defense signaling through modification of cell wall components that generate mobile immune signals.

• Specialized pathogen interactions: While BXL4 functions in defense against necrotrophic pathogens , BGAL17 might have evolved specificity for different pathogen types (e.g., biotrophic fungi, bacteria, or oomycetes).

• Intersection with abiotic stress responses: Cell wall remodeling often serves dual roles in biotic and abiotic stress responses. BGAL17 might function at this intersection, potentially explaining why its role hasn't been clearly identified in single-stress studies.

Research approaches should include pathogen challenge assays with multiple pathogen types in bgal17 mutants, hormone response analysis, and investigation of cell wall composition changes in response to infection.

What are the most promising research directions for understanding BGAL17's role in plant development and stress responses?

The most promising research directions for elucidating BGAL17's functions include:

• Comprehensive expression profiling: Generate detailed spatiotemporal maps of BGAL17 expression across development and under various stresses using the BGAL17 Antibody. This would build upon approaches used with BXL4, which showed specific induction patterns during pathogen infection .

• Structure-function analysis: Determine the three-dimensional structure of BGAL17 and identify catalytic residues to compare with related enzymes like BXL1 and BXL4, which possess both xylosidase and arabinosidase activities .

• Cell wall polymer specificity: Determine which cell wall components are modified by BGAL17 through in vitro enzyme assays and in vivo cell wall composition analysis of mutants, similar to analyses conducted for BXL4 .

• Regulatory network mapping: Identify transcription factors controlling BGAL17 expression and compare with known regulators of other cell wall modification enzymes to place BGAL17 in appropriate regulatory networks.

• Metabolic engineering applications: Explore whether manipulation of BGAL17 can improve plant resistance to pathogens, similar to how BXL4 overexpression enhanced resistance to B. cinerea .

Table 3: Priority Research Questions for BGAL17 Functional Characterization

Each of these directions builds upon successful approaches used with related proteins like BXL4, which has demonstrated roles in plant immunity .