At5g34940 Antibody

What is At5g34940 and what is its functional role in plant cells?

At5g34940 encodes a glycoside hydrolase family 79 protein that functions as an endo beta-glucuronidase/heparanase in Arabidopsis thaliana. This enzyme is involved in carbohydrate metabolism and cell wall modification during plant development. Proteomic studies have demonstrated its presence in both early (5-day) and later (11-day) developmental stages of etiolated hypocotyls, with higher abundance observed in younger tissues . The protein participates in cell wall remodeling processes that are critical during cell elongation phases. As part of the glycoside hydrolase superfamily, it catalyzes the hydrolysis of glycosidic bonds between carbohydrates and between carbohydrates and non-carbohydrate moieties, contributing to cell wall plasticity and extensibility during growth.

How does At5g34940 expression change during plant development?

At5g34940 exhibits a developmentally regulated expression pattern. Proteomic analysis of Arabidopsis etiolated hypocotyls has revealed that while At5g34940 is present in both 5-day (actively elongating) and 11-day (post-elongation) stages, its relative abundance decreases significantly in older tissues . This expression pattern suggests a more prominent role during active cell elongation processes. The differential regulation aligns with the typical expression profiles of other cell wall-modifying enzymes that show higher activity during phases of rapid growth. This temporal expression pattern likely reflects the protein's involvement in cell wall loosening and reorganization that facilitates the expansion of young plant cells.

What is the relationship between At5g34940 and other cell wall proteins?

At5g34940 functions within a complex network of cell wall-modifying proteins. Proteomic studies have identified numerous proteins acting on carbohydrates that work in concert during cell elongation, including multiple xyloglucan endotransglucosylase/hydrolases (XTHs), expansins, and other glycoside hydrolases . In particular, the expression profile of At5g34940 shows similarities to other proteins like AtXTH4 and AtXTH33, which also display decreased abundance in 11-day versus 5-day hypocotyls. This coordinated expression suggests functional relationships in the cell wall remodeling machinery. Understanding these relationships is crucial for comprehending the orchestrated process of cell wall modification during plant development.

What are the recommended validation steps for At5g34940 antibodies before experimental use?

Thorough validation of At5g34940 antibodies is essential before conducting experiments. Start with Western blot analysis using both recombinant At5g34940 protein and Arabidopsis protein extracts from tissues known to express the target (particularly 5-day etiolated hypocotyls where expression is highest) . The antibody should detect a single band at the expected molecular weight of the mature protein (approximately 68-70 kDa after post-translational modifications). Perform peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish signal detection. Include appropriate negative controls such as protein extracts from At5g34940 knockout mutants where the band should be absent. Additionally, validate antibody specificity against closely related GH family proteins to ensure no cross-reactivity. Document all validation steps with clear images and quantitative assessments of specificity and sensitivity.

How should optimal conditions for At5g34940 immunolocalization be determined?

Optimizing immunolocalization conditions for At5g34940 requires systematic testing of fixation, permeabilization, and antibody incubation parameters. Begin with testing different fixatives: 4% paraformaldehyde preserves protein antigenicity while maintaining cellular architecture, but glutaraldehyde may provide better ultrastructural preservation at the expense of some epitope masking. For cell wall proteins like At5g34940, enzymatic digestion with pectolyases or cellulases may be necessary to improve antibody accessibility. Test antibody dilutions ranging from 1:100 to 1:1000 to determine optimal signal-to-noise ratio. When imaging, compare patterns in tissues known to have differential expression (5-day versus 11-day hypocotyls) . Always include controls: (1) secondary antibody-only to assess background, (2) pre-immune serum to evaluate non-specific binding, and (3) when possible, tissue from knockout mutants. Document optimization trials in a systematic manner, recording specific conditions and their corresponding outcomes.

What techniques are most effective for detecting post-translational modifications of At5g34940?

Detecting post-translational modifications (PTMs) of At5g34940 requires specialized approaches beyond standard antibody techniques. Phosphorylation can be detected using phospho-specific antibodies generated against predicted phosphorylation sites, coupled with validation by phosphatase treatment of protein samples. For glycosylation analysis, treat protein extracts with glycosidases (PNGase F for N-linked glycans or O-glycosidase for O-linked glycans) before Western blotting to observe mobility shifts. Mass spectrometry provides comprehensive PTM identification: perform immunoprecipitation with At5g34940 antibodies followed by LC-MS/MS analysis. Different extraction protocols may be necessary to preserve specific modifications; for example, phosphorylation studies require phosphatase inhibitors during extraction. Compare PTM patterns between different developmental stages, as modifications may contribute to the functional changes observed between 5-day and 11-day hypocotyls . Create a systematic catalog of identified modifications correlated with developmental stages and cellular conditions.

How can I design experiments to investigate At5g34940's role in cell wall remodeling during elongation?

To investigate At5g34940's role in cell wall remodeling, implement a multi-faceted experimental approach. First, establish a developmental time course using Arabidopsis hypocotyls from 3, 5, 7, 9, and 11 days post-germination to expand upon existing data showing differential expression between days 5 and 11 . Apply immunolocalization with At5g34940 antibodies to map spatial distribution changes throughout development. Complement this with gene expression analysis using qRT-PCR and in situ hybridization. Generate knockout and overexpression lines to assess phenotypic consequences on hypocotyl elongation rates, cell wall composition, and mechanical properties. For detailed functional characterization, employ enzymatic assays using protein extracted from these transgenic lines to measure endo beta-glucuronidase activity against various substrates. Utilize Atomic Force Microscopy to assess changes in cell wall elasticity. Combine these approaches with comprehensive cell wall composition analysis using techniques like Comprehensive Microarray Polymer Profiling (CoMPP) to correlate At5g34940 levels with specific structural changes in cell wall polysaccharides during the elongation period.

What controls should be included when using At5g34940 antibodies for Western blot analysis?

Robust Western blot analysis with At5g34940 antibodies requires comprehensive controls to ensure reliable data interpretation. Include the following controls: (1) Positive control: recombinant At5g34940 protein or protein extract from tissues with confirmed high expression (5-day etiolated hypocotyls) ; (2) Negative controls: protein extract from At5g34940 knockout mutants and from tissues with minimal expression; (3) Loading controls: probing for constitutively expressed proteins like actin or tubulin, or total protein staining with Ponceau S; (4) Specificity controls: pre-incubation of antibody with immunizing peptide to demonstrate specific binding; (5) Related protein controls: testing against recombinant proteins from other GH family members to assess cross-reactivity potential. For developmental studies, include extracts from multiple time points (e.g., 5-day and 11-day samples) to confirm the expected expression pattern . Document detailed experimental conditions: extraction buffer composition, protein quantification method, sample preparation procedures, gel percentage, transfer conditions, blocking reagents, antibody dilutions, incubation times and temperatures, and detection method specifications.

How can co-immunoprecipitation be optimized to study At5g34940 protein interactions?

Optimizing co-immunoprecipitation (co-IP) for At5g34940 requires careful consideration of plant cell wall protein extraction challenges. Begin with buffer optimization: test multiple extraction buffers containing different detergents (0.5-1% NP-40, Triton X-100, or digitonin) to solubilize membrane-associated proteins while preserving protein-protein interactions. Include protease inhibitors and maintain cold conditions throughout. For crosslinking approaches, test both formaldehyde (0.5-1%) and DSP (dithiobis(succinimidyl propionate)) at various concentrations and incubation times. Compare protein extraction from 5-day hypocotyls (higher At5g34940 expression) versus 11-day hypocotyls to identify developmental stage-specific interaction partners. For the IP step, test both direct antibody conjugation to beads and protein A/G approaches to determine which provides better signal-to-noise ratio. Include appropriate controls: (1) non-immune IgG precipitation, (2) samples from knockout plants, and (3) reciprocal co-IPs with antibodies against suspected interaction partners. Validate interactions using alternative methods such as split-GFP or FRET in planta. Create a detailed protocol documenting optimized conditions to ensure reproducibility.

Why might At5g34940 antibody sensitivity vary between different plant tissues and how can this be addressed?

Variation in At5g34940 antibody sensitivity across plant tissues stems from multiple factors requiring systematic troubleshooting. Cell wall composition differences can impede antibody accessibility, particularly in tissues with extensive secondary cell wall development. To address this, optimize tissue-specific extraction protocols: for recalcitrant tissues, test sequential extraction with increasingly stringent buffers, longer extraction times, or additional mechanical disruption steps. Consider using cell wall degrading enzymes as pretreatment, but carefully optimize concentrations to avoid epitope destruction. The presence of secondary metabolites in certain tissues can interfere with antibody binding; incorporate PVPP (polyvinylpolypyrrolidone) or specific removal steps targeting phenolics or terpenoids. Differential post-translational modifications between tissues may affect epitope recognition; generate multiple antibodies targeting different protein regions or use denaturing conditions that expose core epitopes. For comparative studies between tissues, develop tissue-specific standard curves using recombinant At5g34940 protein spiked into extract from knockout plants. Document tissue-specific protocol modifications and their effects on detection sensitivity to create a comprehensive reference for future experiments.

How can I resolve inconsistent results when comparing At5g34940 protein levels between developmental stages?

Resolving inconsistencies in At5g34940 detection between developmental stages requires addressing technical and biological variables systematically. First, standardize sample collection timing precisely, as studies show significant abundance differences between 5-day and 11-day hypocotyls . Develop stage-specific extraction protocols that account for differences in cell wall composition and protein extractability. For quantitative Western blots, implement batch processing of samples from different stages and include internal loading controls targeting stable reference proteins or total protein normalization. Consider extractability differences: perform sequential extractions with buffers of increasing stringency and analyze each fraction separately. To address biological variability, increase biological replicates (minimum n=6) and ensure growth conditions are precisely controlled across experiments. Perform spike-in recovery experiments with recombinant At5g34940 protein to quantify extraction efficiency across stages. For absolute quantification, develop a quantitative ELISA or implement stable isotope-labeled peptide standards for targeted proteomics approaches. Document complete methodological details, including growth conditions, harvesting times, and extraction protocols, to facilitate reproducibility across different laboratories.

What advanced proteomic approaches can enhance detection and characterization of At5g34940 in complex samples?

Advanced proteomic approaches offer sophisticated solutions for At5g34940 detection and characterization challenges. Implement targeted proteomics using Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) with isotopically labeled peptide standards derived from unique At5g34940 regions. This approach provides absolute quantification even in complex samples and allows detection of specific proteoforms. For comprehensive PTM mapping, combine enrichment strategies (phosphopeptide enrichment, glycopeptide capture) with high-resolution mass spectrometry. Apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe structural dynamics and identify regions involved in substrate binding or protein-protein interactions. For spatial information within tissue contexts, implement advanced imaging mass spectrometry techniques or proximity labeling approaches like BioID or APEX2 fused to At5g34940. To capture the dynamic cell wall proteome comprehensively, apply pulse-chase SILAC (Stable Isotope Labeling with Amino acids in Cell culture) adapted for plant systems to track protein turnover rates across developmental stages like the 5-day to 11-day transition observed in previous studies . Combine these techniques with sophisticated computational algorithms for integrated multi-omics analysis to place At5g34940 function within broader cellular networks.

How should changes in At5g34940 abundance be interpreted in the context of cell elongation?

Interpreting changes in At5g34940 abundance requires integration with cellular and physiological contexts. The observed higher abundance in 5-day versus 11-day hypocotyls correlates with active cell elongation phases, suggesting a functional role in promoting growth. Analyze this pattern alongside other cell wall-modifying enzymes showing similar expression profiles, such as specific XTHs (AtXTH4, AtXTH33) and expansins, to identify coordinated functional modules. When interpreting abundance changes, distinguish between transcriptional regulation and post-translational mechanisms by comparing protein levels with transcript data. Consider spatial distribution within tissues, as localized accumulation may indicate specific action sites despite moderate changes in total abundance. The decrease in At5g34940 between days 5 and 11 may reflect transition from primary to secondary wall formation. To properly interpret abundance changes, correlate them with measurable cell wall properties (extensibility, polymer composition) and growth parameters across the same timepoints. Create integrated models that connect observed abundance patterns with specific phases of the cell growth cycle and mechanical properties of the expanding cell wall.

What approaches can detect potential functional redundancy between At5g34940 and related glycoside hydrolases?

Detecting functional redundancy between At5g34940 and related glycoside hydrolases requires complementary genetic and biochemical approaches. Begin with comprehensive phylogenetic analysis to identify closely related GH family members expressed in similar tissues. Generate single knockouts for At5g34940 and related genes, followed by systematic creation of double, triple, and higher-order mutants to identify enhanced or emergent phenotypes indicating redundancy. Perform detailed growth analyses under normal and stress conditions, measuring parameters like hypocotyl elongation rates, cell size distributions, and cell wall mechanical properties. Utilize CRISPR-Cas9 to create tissue-specific knockouts when global mutations are lethal. Complement genetic approaches with biochemical substrate specificity assays comparing recombinant proteins on defined substrates and native cell wall preparations. Perform transcriptome analysis on single and higher-order mutants to identify compensatory gene expression changes. Implement protein-protein interaction studies to determine if related GHs participate in the same protein complexes. Document all observed compensatory mechanisms between related glycoside hydrolases with quantitative metrics of functional overlap to establish a detailed redundancy map within this important cell wall enzyme family.

How can At5g34940 antibodies be used to investigate protein behavior under abiotic stress conditions?

At5g34940 antibodies can be leveraged to investigate protein behavior under abiotic stress through systematic experimental designs. Establish stress response profiles by subjecting Arabidopsis seedlings to graduated levels of drought, heat, salt, and oxidative stress, then quantify At5g34940 protein levels via Western blot at multiple timepoints post-stress induction. Compare these patterns with other cell wall proteins known to respond to stress, including peroxidases and berberine bridge enzymes that show stress-specific expression patterns . For spatial analysis, perform immunolocalization studies before and after stress treatment to detect potential relocalization within tissues or cells. Investigate post-translational modifications induced by stress using phospho-specific antibodies or mass spectrometry analysis of immunoprecipitated protein. Combine these approaches with cell wall composition analysis to correlate At5g34940 behavior with specific structural adaptations under stress. For functional validation, compare wild-type and At5g34940 knockout responses to various stresses, measuring growth parameters, survival rates, and cell wall integrity. Integrate these data with transcriptomic analysis to place At5g34940 within specific stress response networks and pathways, creating a comprehensive model of its role in stress adaptation.