At5g44940 Antibody

What is the At5g44940 protein and what biological functions does it serve in Arabidopsis thaliana?

At5g44940 (UniProt: Q9FLA3) is a protein expressed in Arabidopsis thaliana (Mouse-ear cress), a model organism extensively used in plant molecular biology research. While the full characterization of this protein continues to evolve through ongoing research, it plays roles in plant cellular processes. The protein has been studied in contexts of plant development and stress responses, making antibodies against it valuable for investigating fundamental plant biology mechanisms .

When designing experiments to investigate At5g44940 function, researchers should consider its expression patterns across different tissue types and developmental stages. Temporal expression analysis often reveals critical insights into protein function that static analyses might miss.

What are the key specifications of commercially available At5g44940 antibodies?

The At5g44940 antibody is typically available as a rabbit-derived polyclonal antibody designed specifically for research applications. The typical specifications include:

These antibodies are specifically developed for research purposes only and are not intended for diagnostic or therapeutic applications .

What is the recommended sample preparation protocol for plant tissues when using At5g44940 antibody?

Effective sample preparation is critical for successful detection of At5g44940 in plant tissues. The following methodological approach is recommended:

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

• Grind the frozen tissue to a fine powder using a pre-chilled mortar and pestle, maintaining frozen conditions.

• Add extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with freshly added protease inhibitors (1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin A).

• Use 3-5 ml of buffer per gram of tissue and homogenize thoroughly.

• Centrifuge the homogenate at 12,000 × g for 15 minutes at 4°C.

• Collect the supernatant and determine protein concentration using Bradford or BCA assay.

• Prepare aliquots and store at -80°C to avoid repeated freeze-thaw cycles.

When working with difficult tissues, consider adding 5% polyvinylpolypyrrolidone (PVPP) to your extraction buffer to remove interfering phenolic compounds often present in plant tissues.

What are the optimal western blotting conditions for At5g44940 antibody?

For optimal western blot results using the At5g44940 antibody, follow this methodological approach:

Critical considerations include using freshly prepared samples, including appropriate positive and negative controls, and optimizing antibody dilutions for each new lot of antibody. If background is high, consider using a different blocking agent like 3-5% BSA instead of milk, or increasing the washing steps (5 washes, 5 minutes each with TBST) .

How can I validate the specificity of At5g44940 antibody in my experimental system?

Validating antibody specificity is essential for reliable research outcomes. Implement these methodological approaches:

• Genetic Controls: Compare wild-type plants with At5g44940 knockout/knockdown mutants. The antibody should show significantly reduced or absent signal in the mutant samples.

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (10-100× molar excess) before applying to the sample. Specific signals should be blocked or significantly reduced.

• Recombinant Protein Controls: Use purified recombinant At5g44940 protein as a positive control to verify the correct molecular weight detection.

• Multiple Detection Methods: Confirm results using at least two different techniques (e.g., western blot and immunofluorescence).

• Tissue-specific Expression Analysis: Compare antibody signals in tissues known to have different expression levels of At5g44940 based on transcriptomic data.

• Mass Spectrometry Validation: Perform immunoprecipitation followed by mass spectrometry to confirm that the precipitated protein is indeed At5g44940.

These validation steps should be documented thoroughly in your research records and included in any publications to enhance reproducibility .

What are the recommended immunohistochemistry protocols for At5g44940 antibody in plant tissues?

Immunohistochemistry with plant tissues requires specialized procedures due to the cell wall and other unique plant cellular components. Follow this optimized protocol:

• Fixation: Fix fresh plant tissue in 4% paraformaldehyde in PBS overnight at 4°C.

• Tissue Processing:

  • Dehydrate through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%, 100%)

  • Clear with xylene or xylene substitute

  • Infiltrate and embed in paraffin

• Sectioning: Cut 5-8 μm thick sections and mount on positively charged slides.

• Deparaffinization and Rehydration:

  • Xylene (2 × 10 min)

  • 100% ethanol (2 × 5 min)

  • 95%, 85%, 70%, 50% ethanol (3 min each)

  • Distilled water (5 min)

• Antigen Retrieval: Incubate slides in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 minutes.

• Blocking: 5% normal goat serum, 0.3% Triton X-100 in PBS for 1 hour at room temperature.

• Primary Antibody: Apply At5g44940 antibody at 1:100-1:200 dilution in 1% BSA, 0.3% Triton X-100 in PBS overnight at 4°C.

• Washing: PBS (3 × 5 min).

• Secondary Antibody: Apply fluorophore-conjugated or enzyme-linked secondary antibody at recommended dilution for 1-2 hours at room temperature.

• Final Washing: PBS (3 × 10 min).

• Counterstaining and Mounting: Use DAPI for nuclear staining and mount with anti-fade mounting medium.

For best results, always include both positive and negative controls, and consider using an antigen retrieval step if signal strength is weak .

What are common troubleshooting strategies for weak or inconsistent signals with At5g44940 antibody?

When encountering weak or inconsistent signals when using the At5g44940 antibody, implement these methodological troubleshooting strategies:

Additionally, consider that At5g44940 might be subject to developmental or stress-induced regulation. Document the precise growth conditions, developmental stage, and treatment conditions for all samples to ensure reproducibility .

How can I design experiments to study At5g44940 protein interactions using co-immunoprecipitation?

To effectively study protein interactions involving At5g44940 using co-immunoprecipitation (Co-IP), follow this methodological framework:

• Sample Preparation:

  • Extract proteins using a gentle lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA) supplemented with protease inhibitors.

  • Clear lysate by centrifugation at 14,000 × g for 15 minutes at 4°C.

  • Pre-clear with Protein A/G beads to reduce non-specific binding.

• Antibody Binding:

  • Incubate 1-5 μg of At5g44940 antibody with 500-1000 μg of protein extract overnight at 4°C with gentle rotation.

  • Add 30-50 μl of pre-washed Protein A/G magnetic beads and incubate for 3-4 hours at 4°C.

• Washing and Elution:

  • Wash beads 4-5 times with wash buffer (lysis buffer with reduced detergent).

  • Elute bound proteins with 2X SDS sample buffer at 95°C for 5 minutes.

• Controls:

  • Critical control: Perform parallel IP with pre-immune serum or IgG from the same species.

  • Include input sample (5-10% of starting material) on your gel.

  • Consider reciprocal Co-IP with antibodies against suspected interacting partners.

• Detection Methods:

  • Western blotting with antibodies against suspected interacting partners.

  • Mass spectrometry analysis for unbiased discovery of novel interactors.

• Validation:

  • Confirm interactions using orthogonal methods such as yeast two-hybrid, FRET, or bimolecular fluorescence complementation.

  • Use genetic approaches (double mutants, overexpression) to validate functional significance.

This approach allows for the identification of protein complexes involving At5g44940, providing insights into its functional roles within plant cellular networks .

What considerations are important when analyzing post-translational modifications of At5g44940?

Post-translational modifications (PTMs) can significantly impact protein function, localization, and stability. When investigating PTMs of At5g44940, consider these methodological approaches:

• Prediction and Targeting:

  • Use bioinformatic tools to predict potential PTM sites (phosphorylation, glycosylation, ubiquitination, etc.).

  • Design experiments to target these specific modifications.

• Sample Preparation:

  • Include appropriate PTM-preserving inhibitors in extraction buffers (phosphatase inhibitors for phosphorylation studies, deubiquitinase inhibitors for ubiquitination studies).

  • Consider enrichment techniques specific to the PTM of interest (phosphopeptide enrichment, ubiquitin remnant motif antibodies).

• Detection Methods:

  • Western Blotting: Use modification-specific antibodies (anti-phospho, anti-ubiquitin) alongside At5g44940 antibody.

  • Mass Spectrometry: Perform MS/MS analysis after enrichment for specific PTMs.

  • Mobility Shift Assays: Detect changes in protein migration following treatments that affect PTMs.

• Validation Strategies:

  • Use kinase/phosphatase inhibitors to manipulate phosphorylation status.

  • Generate site-specific mutants (e.g., serine to alanine) to prevent specific modifications.

  • Perform in vitro enzymatic assays to confirm modification sites.

Understanding At5g44940's post-translational modifications can provide crucial insights into its regulation and function under different environmental conditions or developmental stages .

How can At5g44940 antibody be utilized in chromatin immunoprecipitation (ChIP) studies?

While At5g44940 may not be a conventional target for ChIP studies unless it has DNA-binding properties, adapting the antibody for chromatin immunoprecipitation requires specific methodological considerations:

• Cross-linking and Chromatin Preparation:

  • Cross-link plant tissue with 1% formaldehyde for 10-15 minutes under vacuum.

  • Quench with 0.125 M glycine for 5 minutes.

  • Extract nuclei using a buffer containing 0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 1% Triton X-100, 5 mM β-mercaptoethanol, and protease inhibitors.

  • Sonicate to achieve chromatin fragments of 200-500 bp.

• Immunoprecipitation Optimization:

  • Pre-clear chromatin with Protein A/G beads for 1-2 hours.

  • Use 5-10 μg of At5g44940 antibody per IP reaction.

  • Incubate overnight at 4°C with rotation.

  • Include appropriate controls: IgG negative control and a positive control antibody against a known DNA-binding protein.

• Washing and Elution:

  • Perform stringent washing to remove non-specific interactions.

  • Recommended wash buffers: Low salt (150 mM NaCl), High salt (500 mM NaCl), LiCl wash (250 mM LiCl), and TE buffer.

  • Elute bound complexes with elution buffer (1% SDS, 0.1 M NaHCO₃).

• Reverse Cross-linking and DNA Purification:

  • Reverse cross-links by incubating at 65°C overnight.

  • Treat with RNase A and Proteinase K.

  • Purify DNA using phenol-chloroform extraction or commercial kits.

• Analysis Options:

  • qPCR targeting specific genomic regions of interest.

  • ChIP-seq for genome-wide binding profile analysis.

If At5g44940 is involved in chromatin-associated processes either directly or as part of a complex, this approach can reveal its genomic targets and provide insights into its regulatory functions .

What are the comparative advantages of protein detection methods when working with At5g44940?

Different protein detection methods offer distinct advantages when studying At5g44940. Understanding these differences helps in selecting the most appropriate technique for specific research questions:

When selecting a method, consider:

• The primary research question (abundance, localization, interaction, modification).

• The expected abundance of At5g44940 in your sample.

• The need for quantitative versus qualitative data.

• Available equipment and expertise.

For initial characterization of At5g44940 expression patterns, a combination of western blotting (for verification of specificity) and immunofluorescence (for localization) offers a comprehensive approach .

How should researchers interpret At5g44940 expression patterns across different plant developmental stages?

Interpreting At5g44940 expression patterns across developmental stages requires careful methodological and analytical considerations:

• Experimental Design Considerations:

  • Include multiple time points spanning key developmental transitions.

  • Use a standardized sampling protocol (same time of day, same position on plant).

  • Collect samples from multiple biological replicates (minimum n=3).

  • Consider both vegetative and reproductive tissues.

• Quantification Approaches:

  • Perform densitometry analysis of western blots with normalization to stable reference proteins (e.g., actin, tubulin, or GAPDH).

  • Use technical replicates to ensure measurement reliability.

  • Consider relative quantification across developmental stages rather than absolute values.

• Correlation Analysis:

  • Compare protein expression data with available transcriptomic data for At5g44940.

  • Look for discrepancies that might indicate post-transcriptional regulation.

  • Correlate expression patterns with known developmental processes or environmental responses.

• Visualization and Statistical Analysis:

  • Create developmental expression profile graphs with error bars.

  • Perform statistical tests appropriate for time-series data (e.g., repeated measures ANOVA).

  • Use clustering methods to identify co-regulated proteins if studying multiple targets.

Changes in expression patterns might indicate functional roles in specific developmental transitions. Correlate these changes with known developmental markers to contextualize At5g44940's potential functions within plant developmental programs .

What are the considerations for using At5g44940 antibody in cross-species studies?

When applying the At5g44940 antibody to study related proteins in other plant species, consider these methodological approaches:

• Sequence Homology Analysis:

  • Perform BLAST searches to identify homologs in target species.

  • Align protein sequences focusing on the epitope region recognized by the antibody.

  • Calculate percent identity and similarity, particularly in the antibody binding region.

• Epitope Conservation Assessment:

  • If the epitope sequence is known, compare it directly across species.

  • Predict antigenicity and surface accessibility of the homologous regions.

  • Consider structural conservation rather than just sequence identity.

• Experimental Validation:

  • Perform western blot titration series with protein extracts from different species.

  • Include positive control (Arabidopsis thaliana) and negative control (species with low homology).

  • Consider using recombinant proteins from target species as standards.

• Optimization for Cross-Species Application:

  • Adjust antibody concentration (often higher concentrations are needed).

  • Modify blocking conditions to reduce background.

  • Optimize incubation times (typically longer incubations).

  • Consider less stringent washing conditions.

• Confirmation Strategies:

  • Verify results with genetic approaches when possible.

  • Perform peptide competition assays with peptides from both Arabidopsis and the target species.

  • Consider mass spectrometry validation of detected proteins.

Cross-species applications can provide valuable evolutionary insights into protein conservation and divergence, but require careful validation to ensure specificity and proper interpretation of results .

How can researchers integrate At5g44940 antibody data with transcriptomics and proteomics studies?

Integrating At5g44940 antibody-based data with -omics approaches provides comprehensive insights into protein function. Follow these methodological strategies:

• Correlation Analysis with Transcriptomics:

  • Compare protein abundance (western blot) with mRNA levels (RNA-seq, microarray) across conditions.

  • Calculate Pearson or Spearman correlation coefficients between protein and transcript levels.

  • Investigate discrepancies that may indicate post-transcriptional regulation.

• Integration with Proteomics Data:

  • Use At5g44940 antibody for immunoprecipitation followed by mass spectrometry (IP-MS).

  • Compare targeted antibody results with global proteomics data.

  • Develop a protein interaction network centered on At5g44940.

• Multi-omics Data Visualization:

  • Create integrated heatmaps showing transcript levels, protein abundance, and post-translational modifications.

  • Use principal component analysis to visualize how At5g44940 behaves relative to other proteins/genes.

  • Develop pathway maps incorporating multiple data types.

• Functional Validation Approaches:

  • Design experiments to test hypotheses generated from integrated analyses.

  • Use genetic approaches (mutants, overexpression lines) to validate predictions.

  • Apply network analysis algorithms to identify functional modules.

This integrated approach helps place At5g44940 within the broader cellular context, revealing its position in regulatory networks and metabolic pathways that may not be apparent from single-technique studies .

What are the best practices for quantitative analysis of At5g44940 in plant stress response studies?

When investigating At5g44940's role in plant stress responses, employ these methodological approaches for rigorous quantitative analysis:

• Experimental Design Considerations:

  • Include appropriate time course sampling (early, middle, late responses).

  • Apply controlled, reproducible stress conditions.

  • Include multiple biological replicates (minimum n=5 for stress studies).

  • Consider dose-response relationships for chemical stressors.

• Quantification Methods:

  • Use quantitative western blotting with fluorescent secondary antibodies for wider dynamic range.

  • Implement internal loading controls resistant to the specific stress (validate stability under your conditions).

  • Consider ELISA for high-throughput quantification across many samples.

• Data Normalization Strategies:

  • Normalize to total protein (Ponceau S, SYPRO Ruby) rather than individual housekeeping proteins.

  • Use multiple reference proteins when possible.

  • Consider band intensity ratios to an unchanging protein as an alternative approach.

• Statistical Analysis Framework:

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

  • Calculate effect sizes, not just statistical significance.

  • Consider non-parametric methods if data doesn't meet normality assumptions.

  • Use time-series specific methods for kinetic analyses.

• Visualization Standards:

  • Present both representative images and quantification graphs.

  • Always include error bars representing biological variation.

  • Use consistent scaling across comparable experiments.

This structured approach ensures reliable quantification of At5g44940 protein dynamics during stress responses, facilitating the discovery of its functional roles in plant stress adaptation mechanisms .

What are the current limitations in At5g44940 antibody research and future directions?

Current research using At5g44940 antibodies faces several methodological limitations, while emerging technologies offer promising future directions:

Current Limitations:

• Antibody Specificity Challenges:

  • Polyclonal antibodies may recognize multiple epitopes with variable affinity.

  • Batch-to-batch variation can affect reproducibility.

  • Cross-reactivity with related proteins may confound results.

• Technical Constraints:

  • Long lead times for antibody production (14-16 weeks) delay research timelines .

  • Limited availability of monoclonal antibodies for At5g44940.

  • Challenges in detecting low-abundance or transiently expressed forms.

• Knowledge Gaps:

  • Incomplete characterization of At5g44940's post-translational modifications.

  • Limited understanding of protein-protein interaction networks.

  • Sparse information on subcellular localization dynamics.

Future Directions:

• Advanced Antibody Technologies:

  • Development of recombinant antibodies with defined epitope binding.

  • Single-domain antibodies (nanobodies) for improved penetration in plant tissues.

  • Antibody engineering for specific post-translational modification detection.

• Integration of New Methodologies:

  • Proximity labeling approaches (BioID, TurboID) to map protein neighborhoods.

  • Super-resolution microscopy for precise subcellular localization.

  • CRISPR-based tagging for endogenous protein tracking.

• Systems Biology Approaches:

  • Multi-omics integration to place At5g44940 in regulatory networks.

  • Mathematical modeling of protein dynamics across developmental stages.

  • Comparative analyses across plant species to understand evolutionary conservation.

• Functional Characterization:

  • Structure-function studies to identify critical protein domains.

  • Genetic engineering to create modified variants for mechanistic studies.

  • Investigation of At5g44940's role in emerging areas of plant biology.

Addressing these limitations and embracing new technologies will advance our understanding of At5g44940's functions in plant biology and potentially reveal novel applications in agricultural biotechnology .

How should researchers document and report At5g44940 antibody validation for publication?

Thorough documentation and reporting of antibody validation are essential for research reproducibility. Follow these methodological best practices when preparing manuscripts involving At5g44940 antibody:

• Essential Information to Report:

  • Complete antibody information: source, catalog number, lot number, RRID if available.

  • Host species, clonality (polyclonal/monoclonal), and immunogen details.

  • Working dilutions for each application with optimization details.

  • Storage and handling conditions that maintained antibody performance.

• Validation Methods Documentation:

  • Include images of complete western blots showing molecular weight markers and all bands.

  • Document positive and negative controls (genetic knockouts, competing peptides).

  • Report reproducibility assessments across multiple biological replicates.

  • Quantify signal-to-noise ratios where applicable.

• Experimental Protocol Reporting:

  • Provide detailed protocols including buffer compositions and incubation conditions.

  • Report sample preparation methods thoroughly, including protein extraction protocols.

  • Document image acquisition parameters (exposure times, gain settings).

  • Share quantification methods and statistical approaches.

• Addressing Limitations Transparently:

  • Acknowledge any cross-reactivity observed in validation studies.

  • Discuss batch-to-batch variation if multiple antibody lots were used.

  • Note any inconsistencies or unexpected results during validation.

  • Address how experimental conditions might affect antibody performance.

• Data Sharing Considerations:

  • Deposit raw image files in appropriate repositories when possible.

  • Consider providing validation data as supplementary material.

  • Share detailed protocols through platforms like protocols.io.

  • Make validation reagents available to the research community when feasible.