BGLU41 Antibody

What is BGLU41 and what is its biological significance?

BGLU41 (Beta-Glucosidase 41) is a protein encoded by the AT5G54570 gene in Arabidopsis thaliana, commonly known as mouse-ear cress. It belongs to the beta-glucosidase family of enzymes that catalyze the hydrolysis of glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides. These enzymes play crucial roles in various plant physiological processes including plant defense responses, cell wall modification, phytohormone activation, and lignification. The protein is characterized by its UniProt accession number Q9FIU7 and is part of the larger glycoside hydrolase family . Understanding BGLU41's function provides insights into plant metabolism, development, and stress responses, making it a valuable target for agricultural and biotechnological research.

What types of BGLU41 antibodies are available for research?

Based on available information, BGLU41 antibodies are commercially available from scientific suppliers in different formats. The antibody with catalog code CSB-PA863768XA01DOA is specifically raised against Arabidopsis thaliana BGLU41 protein and is available in two size options: 0.1ml and 2ml . These antibodies are likely polyclonal antibodies generated in rabbits or other host animals. While the search results don't specify the exact types available, researchers should be aware that antibodies against plant proteins typically come in several varieties:

• Polyclonal antibodies - recognize multiple epitopes and offer high sensitivity

• Monoclonal antibodies - recognize a single epitope for higher specificity

• Recombinant antibodies - engineered for consistent performance

Each type has distinct advantages depending on the intended application, with polyclonal antibodies generally offering better sensitivity while monoclonal antibodies provide higher specificity for particular epitopes.

What are the primary applications for BGLU41 antibody in plant research?

BGLU41 antibodies are versatile tools in plant molecular biology research with several key applications. They can be used for western blotting to detect and quantify BGLU41 protein expression levels in plant tissue extracts, revealing information about protein abundance under different experimental conditions. Immunoprecipitation techniques allow researchers to isolate BGLU41 and its interacting partners from complex protein mixtures, helping to elucidate protein-protein interactions and regulatory networks. In immunohistochemistry and immunofluorescence microscopy, these antibodies enable visualization of BGLU41's subcellular localization and tissue distribution patterns, providing spatial information about the protein's function. Additionally, BGLU41 antibodies can be utilized in ELISA assays for quantitative detection of the protein in plant samples. These diverse applications make BGLU41 antibodies essential for comprehensive studies of beta-glucosidase activity in Arabidopsis and comparative analyses with other plant species.

What is the recommended protocol for western blotting with BGLU41 antibody?

For optimal western blotting results with BGLU41 antibody, researchers should follow a protocol similar to those used for other plant proteins. Begin with proper sample preparation by extracting proteins from Arabidopsis tissues using a buffer containing 100 mM Tris-HCl (pH 7.4), 150 mM NaCl, appropriate protease inhibitors, and phosphatase inhibitors such as Na3VO4 if phosphorylation status is important . After homogenization, centrifuge the samples and collect the supernatant containing soluble proteins.

Separate proteins using SDS-PAGE (10-12% gels are typically suitable for BGLU41), then transfer to nitrocellulose membranes using standard methods. Block membranes with 5% skim milk in washing buffer (100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% Tween 20) for 1 hour at room temperature . Incubate with BGLU41 primary antibody (1:1000 to 1:2000 dilution is recommended as a starting point) overnight at 4°C. After washing three times with washing buffer, incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.

Develop using enhanced chemiluminescence and expose to X-ray film or detect using an imaging system . BGLU41 should appear at approximately 60-65 kDa, though exact size may vary depending on post-translational modifications. Always include appropriate positive and negative controls to validate antibody specificity.

How should BGLU41 antibody be validated before experimental use?

Thorough validation of BGLU41 antibody is essential before conducting experiments to ensure reliable results. Begin with a basic western blot using wild-type Arabidopsis thaliana extracts alongside a bglu41 knockout/knockdown mutant line as a negative control. This comparison will confirm antibody specificity by demonstrating the absence or reduction of the target band in the mutant sample. Additionally, preabsorption tests can be performed by incubating the antibody with purified BGLU41 protein prior to immunoblotting, which should eliminate or significantly reduce signal if the antibody is specific.

For more rigorous validation, consider performing immunoprecipitation followed by mass spectrometry to confirm that the antibody is capturing the intended target. Cross-reactivity testing with other members of the beta-glucosidase family, particularly the closely related BGLU40 and BGLU42, will help establish the specificity range of the antibody. Carefully document all validation steps, including antibody dilutions, incubation conditions, and detection methods, to ensure reproducibility of results.

Researchers should also evaluate lot-to-lot variation when using commercial antibodies by testing new lots against previously validated batches to ensure consistent performance across experiments.

What are the optimal conditions for immunohistochemistry with BGLU41 antibody?

For successful immunohistochemistry using BGLU41 antibody in Arabidopsis thaliana tissues, careful sample preparation and optimized staining conditions are essential. Begin by fixing plant tissues in 4% paraformaldehyde for 2-4 hours, followed by embedding in paraffin or resin. Cut sections at 5-8 μm thickness and mount on adhesive slides. Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20-30 minutes to unmask epitopes that may have been altered during fixation.

Block non-specific binding sites with 5% normal serum (from the same species as the secondary antibody) in PBS containing 0.3% Triton X-100 for 1 hour at room temperature. Incubate sections with BGLU41 primary antibody at a 1:100 to 1:500 dilution (starting point for optimization) in blocking buffer overnight at 4°C. After washing with PBS, apply fluorescently-labeled or HRP-conjugated secondary antibody for 1-2 hours at room temperature.

For brightfield microscopy, develop using DAB substrate and counterstain with hematoxylin. For fluorescence visualization, counterstain with DAPI to visualize nuclei. Include controls without primary antibody and, if available, tissues from bglu41 mutant plants. Because plant tissues contain complex cell walls and vacuoles, longer incubation times and higher antibody concentrations may be necessary compared to animal tissues. The expected localization pattern for BGLU41 would include the endoplasmic reticulum, vacuoles, or cell wall regions, depending on its specific function in the plant tissue being examined.

What are common issues when using BGLU41 antibody and how can they be resolved?

When working with BGLU41 antibody, researchers commonly encounter several challenges that require specific troubleshooting approaches. High background signal in western blots or immunostaining is frequently observed and can be mitigated by increasing blocking time, using alternative blocking agents (such as BSA or commercial blockers), increasing wash duration/frequency, or further optimizing antibody dilutions. For western blotting specifically, adding 0.05-0.1% SDS to the washing buffer may help reduce non-specific binding.

Another common issue is weak or absent signal, which may result from insufficient antigen, protein degradation, or ineffective antibody. To address this, researchers should verify protein extraction efficiency, add additional protease inhibitors during sample preparation, and consider using alternative extraction buffers optimized for plant tissues. Signal amplification systems like biotin-streptavidin can enhance detection sensitivity.

For plant samples specifically, endogenous peroxidase activity can interfere with HRP-based detection methods. This can be mitigated by including a hydrogen peroxide treatment step (0.3% H2O2 in methanol for 30 minutes) prior to blocking. Additionally, plant tissues rich in phenolic compounds may cause non-specific antibody binding, which can be reduced by including 0.1% PVPP (polyvinylpolypyrrolidone) in extraction and blocking buffers.

How can BGLU41 antibody be used to study protein-protein interactions?

BGLU41 antibody can be effectively employed to investigate protein-protein interactions through several complementary approaches. Co-immunoprecipitation (Co-IP) is perhaps the most direct method, where BGLU41 antibody is used to pull down the target protein along with its interacting partners from plant cell lysates. For this application, researchers should modify standard immunoprecipitation protocols to use milder lysis conditions (non-ionic detergents like 0.5% NP-40 or 1% Triton X-100) to preserve protein complexes. The precipitated proteins can then be identified using mass spectrometry or western blotting with antibodies against suspected interaction partners.

Proximity ligation assay (PLA) offers another powerful approach for visualizing protein interactions in situ. This technique combines antibody binding with DNA amplification to generate fluorescent signals only when two proteins are in close proximity (< 40 nm). For this application, BGLU41 antibody would be used alongside antibodies against potential interaction partners, each conjugated with different DNA oligonucleotides.

For studying dynamic interactions, researchers can combine BGLU41 immunoprecipitation with crosslinking approaches. Chemical crosslinkers like formaldehyde or DSS (disuccinimidyl suberate) can stabilize transient interactions before cell lysis. Alternatively, photoactivatable crosslinkers allow for precise temporal control of the crosslinking reaction.

How does post-translational modification of BGLU41 affect antibody recognition?

Post-translational modifications (PTMs) of BGLU41 can significantly impact antibody recognition, potentially leading to misleading experimental results if not properly considered. BGLU41, like many plant proteins, may undergo several types of PTMs including glycosylation, phosphorylation, and ubiquitination. These modifications can either mask antibody epitopes or create new recognition sites, affecting the binding efficiency of the antibody.

Glycosylation is particularly relevant for beta-glucosidases like BGLU41, as these enzymes often contain N-linked glycans that can shield peptide epitopes from antibody recognition. If the antibody was raised against a bacterially-expressed recombinant protein (which lacks glycosylation), it might not efficiently recognize the native glycosylated form in plant extracts. To address this issue, researchers can treat samples with deglycosylation enzymes like PNGase F before immunoblotting to remove N-linked glycans and potentially enhance antibody binding.

Phosphorylation states can also alter epitope accessibility. If studying phosphorylation is important, researchers should consider using phosphatase inhibitors such as Na3VO4 during sample preparation, as mentioned in standard western blotting protocols . For comprehensive analysis of how PTMs affect BGLU41 recognition, researchers can compare antibody binding to native protein versus denatured protein, or examine recognition patterns after treatment with various enzymes that remove specific modifications.

When interpreting variable results across different tissues or experimental conditions, researchers should consider whether differences in PTM status rather than protein abundance might explain the observed patterns. For definitive characterization of BGLU41 PTMs, techniques like mass spectrometry following immunoprecipitation can reveal the precise modification landscape and how it correlates with antibody recognition efficiency.

How can BGLU41 antibody be used in conjunction with other techniques to study plant stress responses?

BGLU41 antibody can be integrated with multiple complementary techniques to comprehensively investigate plant stress responses. By combining immunological detection with transcriptomic and metabolomic approaches, researchers can gain insights into how BGLU41 functions within broader stress response networks in Arabidopsis thaliana.

For analyzing spatiotemporal dynamics of BGLU41 expression during stress responses, researchers can combine immunohistochemistry with BGLU41 antibody and live-cell imaging techniques. Time-course experiments exposing plants to various stressors (drought, salinity, pathogen infection) followed by immunoblotting can reveal how BGLU41 protein levels change in response to specific stresses over time. This protein-level data can be correlated with RT-qPCR analysis of BGLU41 transcript levels to understand post-transcriptional regulation mechanisms.

Chromatin immunoprecipitation sequencing (ChIP-seq) using antibodies against transcription factors suspected to regulate BGLU41, combined with BGLU41 protein quantification via western blotting, can help establish regulatory relationships in stress signaling pathways. For understanding BGLU41's enzymatic function during stress, researchers can perform activity assays using artificial substrates like 4-methylumbelliferyl-β-D-glucoside after immunoprecipitating the native protein from stressed and control plants.

Additionally, BGLU41 antibody can be used in conjunction with metabolite profiling to correlate protein abundance with changes in specific metabolites, potentially identifying natural substrates and products. For example, comparing metabolite profiles between wild-type and bglu41 knockout plants under stress conditions, alongside immunological verification of protein abundance, can provide functional insights into BGLU41's role in stress adaptation mechanisms.

What approaches can be used to study BGLU41 localization and trafficking in plant cells?

Investigating the subcellular localization and trafficking of BGLU41 requires a multi-faceted approach combining immunological techniques with advanced microscopy methods. Immunogold electron microscopy using BGLU41 antibody provides the highest resolution visualization of the protein's precise subcellular location, allowing researchers to determine whether it resides in the vacuole, cell wall, endoplasmic reticulum, or other compartments. This technique requires careful fixation and embedding of plant tissues, followed by ultrathin sectioning and immunolabeling with BGLU41 antibody and gold-conjugated secondary antibodies.

Confocal microscopy with immunofluorescence offers another powerful approach, allowing for co-localization studies with organelle-specific markers. By double-labeling with BGLU41 antibody and antibodies against markers for various cellular compartments (such as BiP for ER, γ-TIP for tonoplast, or PIP1 for plasma membrane), researchers can determine BGLU41's primary location and potential trafficking patterns.

For dynamic studies of BGLU41 trafficking, researchers can use pulse-chase experiments combined with immunoprecipitation. By metabolically labeling newly synthesized proteins followed by immunoprecipitation with BGLU41 antibody at different time points, researchers can track the maturation and movement of the protein through cellular compartments. Subcellular fractionation followed by western blotting with BGLU41 antibody provides a complementary biochemical approach to verify microscopy findings.

To study stimulus-induced relocalization, researchers can expose plants to various treatments (hormones, stressors) and examine potential changes in BGLU41 localization using immunofluorescence microscopy. Control experiments should include competitive inhibition with recombinant BGLU41 protein and parallel analysis of bglu41 knockout plants to confirm antibody specificity in localization studies.

How can researchers use BGLU41 antibody to investigate evolutionary conservation across plant species?

BGLU41 antibody presents a valuable tool for comparative studies examining the evolutionary conservation of beta-glucosidases across different plant species. To employ this approach effectively, researchers should first assess the antibody's cross-reactivity with orthologous proteins from diverse plant lineages through western blotting of protein extracts from multiple species. This initial screening will identify which species contain sufficiently conserved epitopes for reliable detection.

For species where cross-reactivity is confirmed, immunohistochemistry can reveal conservation or divergence in tissue-specific expression patterns. This comparative immunolocalization across species can provide insights into functional conservation or specialization of BGLU41 orthologs. Researchers should be mindful that even with cross-reactive antibodies, the intensity of signals may vary between species due to epitope differences rather than protein abundance variations.

Immunoprecipitation followed by mass spectrometry offers a powerful approach for identifying and characterizing BGLU41 orthologs in non-model plants. The precipitated proteins can be analyzed for sequence variations, post-translational modifications, and interaction partners across species. This information can help reconstruct the evolutionary history of beta-glucosidase function in plants and identify conserved functional domains.

For more quantitative evolutionary analysis, researchers can combine immunological detection with structural biology approaches. By comparing the epitope regions recognized by the antibody across species with known crystal structures (or predicted models) of beta-glucosidases, researchers can correlate structural conservation with functional conservation. Additionally, comparing immunological data with genomic and transcriptomic data from multiple species can provide a comprehensive view of how BGLU41 function has evolved throughout plant phylogeny, potentially revealing specialized adaptations in different lineages.

How might BGLU41 antibody be used in plant biotechnology applications?

BGLU41 antibody has significant potential in various plant biotechnology applications, particularly in developing improved crops with enhanced stress tolerance or modified metabolic properties. One promising application is the use of BGLU41 antibody in high-throughput screening platforms to identify plant varieties or transgenic lines with optimal BGLU41 expression levels. Since beta-glucosidases play crucial roles in plant defense mechanisms and hormone activation, plants with optimal BGLU41 expression might demonstrate enhanced resistance to pathogens or improved growth characteristics.

In metabolic engineering efforts, BGLU41 antibody can serve as a monitoring tool to verify protein expression levels in plants engineered to modify specific glycoside hydrolysis pathways. For example, in biofuel research, where efficient breakdown of plant cell walls is desirable, monitoring BGLU41 and related enzyme levels could help optimize biomass conversion processes. Similarly, in plants engineered to produce valuable secondary metabolites, BGLU41 antibody can help track the expression of key enzymes involved in glycoside metabolism.

For agricultural applications focusing on stress tolerance, BGLU41 antibody can be used to correlate protein expression with phenotypic traits across diverse germplasm, potentially identifying naturally occurring variants with superior characteristics. This information could guide traditional breeding programs or genetic engineering approaches aimed at improving crop resilience.

Additionally, BGLU41 antibody could be adapted for the development of immunosensors to monitor plant health in agricultural settings, potentially detecting stress responses before visible symptoms appear. While these applications extend beyond basic research into applied biotechnology, they all build upon the fundamental understanding of BGLU41 function that can be elucidated through careful immunological studies.

What are the challenges in developing improved BGLU41 antibodies for research?

Developing improved BGLU41 antibodies presents several technical challenges that researchers must address to enhance specificity and versatility for advanced applications. One primary challenge is the high sequence similarity between BGLU41 and other members of the beta-glucosidase family in Arabidopsis, which contains over 40 related proteins. This homology makes it difficult to develop highly specific antibodies that don't cross-react with other family members. To overcome this, researchers must carefully select unique epitopes, perhaps from less conserved regions like the C-terminus or specific surface loops, and thoroughly validate specificity against multiple related proteins.

Another significant challenge involves the complex post-translational modifications of plant beta-glucosidases. BGLU41 likely undergoes glycosylation and other modifications that can affect epitope accessibility. Researchers developing new antibodies must consider whether to target modified or unmodified forms of the protein, potentially developing modification-specific antibodies that recognize particular post-translationally modified versions of BGLU41.

The development of monoclonal antibodies against plant proteins presents additional technical hurdles, as plant proteins may be poorly immunogenic in mammalian hosts traditionally used for antibody production. Alternative approaches like phage display technology or recombinant antibody engineering might offer solutions to generate highly specific binding reagents without relying on animal immunization.

For advanced applications like super-resolution microscopy or in vivo imaging, researchers face the challenge of developing antibody fragments or nanobodies that maintain specificity while providing improved tissue penetration and reduced background. These smaller binding reagents may also be more compatible with techniques like expansion microscopy for visualizing BGLU41 localization at nanoscale resolution.

Finally, ensuring reproducibility across different laboratories requires standardized production methods and thorough validation protocols. Recombinant antibody technology offers one solution, providing renewable antibody sources with consistent properties that can be shared between research groups studying BGLU41 and related proteins.

How can multiplexed immunoassays be developed to study BGLU41 in relation to other glycoside hydrolases?

Multiplexed immunoassays represent a powerful approach to simultaneously study BGLU41 alongside other glycoside hydrolases, providing comprehensive insights into their coordinated expression and functional relationships. Developing such assays requires careful antibody selection and validation to ensure specificity and compatibility within a multiplexed format.

For protein microarray-based approaches, researchers can immobilize multiple antibodies against different glycoside hydrolases (including BGLU41) on a single substrate, allowing simultaneous detection of various enzymes from a single plant extract. This approach requires antibodies with similar optimal binding conditions but distinct specificities. Cross-reactivity testing is essential, particularly for closely related beta-glucosidases. The validation process should include testing against recombinant proteins and extracts from knockout plants lacking specific enzymes.

Flow cytometry-based multiplexed assays offer another promising approach, particularly for single-cell analysis. By conjugating antibodies against BGLU41 and other glycoside hydrolases with different fluorophores, researchers can analyze the co-expression patterns of multiple enzymes at the cellular level. This technique requires careful optimization of antibody-fluorophore conjugation to maintain binding specificity while providing distinct fluorescent signals.

Mass cytometry (CyTOF) represents an advanced multiplexing approach where antibodies are labeled with rare earth metals rather than fluorophores, allowing for higher multiplexing capacity without spectral overlap issues. This could enable simultaneous analysis of dozens of different glycoside hydrolases in complex plant samples.

For in situ visualization, multiplexed immunofluorescence techniques like Opal™ multispectral imaging allow sequential staining with multiple antibodies on the same tissue section. This approach can reveal spatial relationships between BGLU41 and other glycoside hydrolases within plant tissues. When developing these assays, researchers must optimize antigen retrieval conditions that work compatibly for all target proteins and include appropriate controls to account for potential antibody cross-reactivity or non-specific binding in complex plant tissues.