At5g38810 Antibody

What is At5g38810 and why is it significant in plant research?

At5g38810 is a gene in Arabidopsis thaliana (mouse-ear cress) that has been identified in studies of plant gene expression and regulation . The significance of this gene lies in its association with LND (Low Nucleosome Density) regions in its promoters, suggesting it may play important roles in transcriptional regulation . The antibody against this protein enables researchers to detect and quantify the At5g38810 protein in various experimental settings, which is crucial for understanding gene function in this model plant organism. Understanding the expression patterns of At5g38810 contributes to our knowledge of plant developmental biology and stress responses.

What are the validated applications for the At5g38810 antibody?

The At5g38810 antibody has been validated for several experimental applications including ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) . These techniques allow researchers to detect and quantify the At5g38810 protein in plant tissue samples. ELISA provides quantitative information about protein expression levels, while Western Blot enables visualization of the protein's molecular weight and assessment of antibody specificity. When designing experiments, researchers should ensure proper controls are included to validate antibody specificity, particularly when working with novel plant tissue types or experimental conditions not previously tested.

What are the optimal storage and handling conditions for the At5g38810 antibody?

For maximum stability and performance, the At5g38810 antibody should be stored at either -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be avoided as they can compromise antibody activity and specificity . The antibody is supplied in liquid form in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . For routine use, aliquoting the antibody into smaller volumes before freezing is recommended to minimize freeze-thaw cycles. When handling the antibody, maintaining sterile technique is essential to prevent microbial contamination, and gloves should be worn to protect both the researcher and the reagent.

How should I design control experiments when using the At5g38810 antibody?

Designing robust controls is essential for validating results with the At5g38810 antibody. A comprehensive control strategy should include:

What is the recommended protocol for using At5g38810 antibody in Western blotting?

For optimal Western blot results with the At5g38810 antibody, follow this methodology:

• Sample Preparation: Extract proteins from Arabidopsis tissues using a buffer containing protease inhibitors to prevent degradation. Quantify protein concentration using Bradford or BCA assay.

• Gel Electrophoresis: Separate 20-40 μg of total protein on a 10-12% SDS-PAGE gel, depending on the predicted molecular weight of At5g38810.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard wet or semi-dry transfer methods.

• Blocking: Block the membrane with 5% non-fat dry milk or BSA in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute the At5g38810 antibody in blocking buffer (recommended dilution range: 1:1000 to 1:2000, optimize for your specific lot) and incubate overnight at 4°C with gentle agitation.

• Washing: Wash the membrane 3-4 times with TBST, 5-10 minutes per wash.

• Secondary Antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000 to 1:10000) in blocking buffer for 1 hour at room temperature.

• Final Washing: Wash 3-4 times with TBST.

• Detection: Visualize using ECL substrate and appropriate imaging system.

This protocol may require optimization based on specific experimental conditions and antibody lot variations. Including appropriate positive and negative controls is essential for result interpretation.

How can I use the At5g38810 antibody to study protein-protein interactions?

The At5g38810 antibody can be effectively employed to investigate protein-protein interactions through several advanced techniques:

• Co-immunoprecipitation (Co-IP): Use the At5g38810 antibody to pull down the target protein along with its interaction partners from plant cell lysates. The precipitated complexes can then be analyzed by mass spectrometry or Western blotting with antibodies against suspected interaction partners.

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ with high sensitivity. Use the At5g38810 antibody in combination with antibodies against potential interaction partners, followed by species-specific secondary antibodies linked to complementary DNA oligonucleotides that, when in close proximity, enable localized DNA amplification and fluorescent detection.

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing): If At5g38810 is suspected to be a transcription factor or chromatin-associated protein, the antibody can be used to identify DNA binding sites genome-wide.

What approaches can be used to validate the specificity of the At5g38810 antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For the At5g38810 antibody, implement these comprehensive validation approaches:

• Genetic Validation:

  • Test the antibody in At5g38810 knockout or knockdown mutants where the protein should be absent or reduced

  • Complement these lines with the wild-type gene and confirm restoration of antibody signal

• Molecular Validation:

  • Overexpress tagged versions of At5g38810 and confirm co-detection with both the antibody and tag-specific antibodies

  • Perform peptide competition assays using the immunizing antigen (recombinant Arabidopsis thaliana At5g38810 protein)

• Analytical Validation:

  • Confirm a single band of appropriate molecular weight in Western blots

  • Verify that immunofluorescence patterns correlate with expected subcellular localization

  • Perform mass spectrometry analysis of immunoprecipitated material to confirm presence of At5g38810

• Cross-Reactivity Assessment:

  • Test the antibody against closely related proteins in the same gene family

  • Evaluate performance in other plant species if sequence homology suggests potential cross-reactivity

These validation steps should be performed prior to using the antibody in critical experiments and should be reported in publications to enhance reproducibility.

What are common issues when using At5g38810 antibody and how can they be resolved?

Researchers frequently encounter several challenges when working with the At5g38810 antibody. Here are common problems and their solutions:

Remember that the At5g38810 antibody has been affinity-purified , which should reduce non-specific binding, but optimization may still be necessary for each experimental system. If problems persist after standard troubleshooting, consider testing a different lot or supplier of the antibody.

How can I optimize the At5g38810 antibody for immunohistochemistry applications?

Although the At5g38810 antibody is primarily validated for ELISA and Western blot applications , it may be adapted for immunohistochemistry (IHC) with appropriate optimization:

• Fixation Protocol: Test multiple fixation methods (4% paraformaldehyde, glutaraldehyde, or combination) to determine which best preserves antigenicity while maintaining tissue morphology. Fixation time should be optimized (typically 2-24 hours depending on tissue thickness).

• Antigen Retrieval: Since fixation can mask epitopes, evaluate different antigen retrieval methods:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 or Tris-EDTA pH 9.0)

  • Enzymatic retrieval (proteinase K, trypsin)

  • No retrieval as a control condition

• Antibody Dilution Series: Test a range of primary antibody dilutions (starting from 1:100 to 1:1000) to determine optimal signal-to-noise ratio.

• Incubation Conditions: Compare overnight incubation at 4°C versus shorter incubations (2-4 hours) at room temperature.

• Detection Systems: Evaluate different secondary antibody systems:

  • Standard indirect detection with fluorescent or enzyme-conjugated secondary antibodies

  • Signal amplification methods (avidin-biotin, tyramide) for low-abundance proteins

• Controls: Include appropriate controls in every experiment:

  • Tissue from knockout/knockdown plants

  • Secondary antibody-only controls

  • Pre-absorption with immunizing peptide

Document all optimization parameters systematically, as the protocol may need to be adjusted for different plant tissues or developmental stages.

How can the At5g38810 antibody be used to investigate transcriptional regulation in plants?

The At5g38810 gene contains LND (Low Nucleosome Density) regions in its promoter , suggesting a potential role in transcriptional regulation. The antibody can be employed to explore this function through several approaches:

• Chromatin Immunoprecipitation (ChIP): If At5g38810 functions as a transcription factor or chromatin-associated protein, ChIP using the antibody can identify DNA binding sites. Combined with next-generation sequencing (ChIP-seq), this approach provides genome-wide mapping of binding locations and potential gene targets.

• Nucleosome Positioning Analysis: The presence of LND regions suggests possible chromatin remodeling functions. The antibody can be used to investigate associations between At5g38810 and chromatin remodeling complexes through co-immunoprecipitation followed by mass spectrometry.

• Transcriptional Response Studies: Correlate At5g38810 protein levels (quantified using the antibody) with expression of potential target genes under various environmental conditions or developmental stages. This can reveal conditions where At5g38810 activity is particularly important.

• Protein Complex Analysis: Use the antibody to isolate native protein complexes containing At5g38810, followed by proteomics analysis to identify interacting partners involved in transcriptional regulation.

Understanding the role of At5g38810 in transcriptional networks may provide insights into plant development and environmental responses, particularly since genes with LND promoter regions often have specialized regulatory functions .

What insights can be gained from studying At5g38810 protein expression across different plant tissues and developmental stages?

Analyzing At5g38810 protein expression patterns using the specific antibody can yield valuable insights into its biological functions:

• Tissue-Specific Expression: The antibody can be used in Western blotting or immunohistochemistry to create a tissue expression map, revealing whether At5g38810 is ubiquitously expressed or restricted to specific plant organs such as roots, young seedlings, leaves, seeds/siliques, floral organs, or stem tissues .

• Developmental Regulation: By sampling tissues at different developmental stages and quantifying protein levels, researchers can determine whether At5g38810 expression is developmentally regulated, suggesting potential roles in specific growth phases.

• Subcellular Localization: Immunofluorescence microscopy with the At5g38810 antibody can reveal the protein's subcellular localization, providing clues about its function. The product information indicates association with the "endomembrane system" , which could be further confirmed and refined.

• Stress-Responsive Expression: Comparing protein levels before and after exposure to various abiotic or biotic stresses can indicate whether At5g38810 participates in stress response pathways.

• Correlation with RNA Expression: Combining protein detection via the antibody with RT-qPCR analysis of mRNA levels can identify potential post-transcriptional regulation mechanisms if protein and mRNA levels are not concordant.

These expression analyses can guide functional studies by identifying conditions or tissues where At5g38810 likely plays important biological roles, informing experimental design for subsequent functional characterization.

How should I quantify and normalize Western blot data when using the At5g38810 antibody?

• Image Acquisition:

  • Capture images within the linear dynamic range of your detection system

  • Avoid saturated pixels which preclude accurate quantification

  • Use the same exposure settings for comparable samples

• Quantification Software:

  • Use specialized software (ImageJ, Image Lab, etc.) to measure band intensity

  • Define consistent regions of interest (ROIs) for all bands and background areas

  • Subtract local background from each band measurement

• Normalization Approaches:

• Statistical Analysis:

  • Perform experiments with at least three biological replicates

  • Use appropriate statistical tests (t-test, ANOVA) based on experimental design

  • Report both normalized values and raw data when possible

• Avoiding Common Pitfalls:

  • Verify that reference protein expression is stable under your experimental conditions

  • Ensure that both target and reference proteins are in the linear range of detection

  • Be cautious when comparing bands across different blots or membranes

What statistical approaches are recommended for analyzing ELISA data with the At5g38810 antibody?

• Standard Curve Analysis:

  • Use purified recombinant At5g38810 protein to generate a standard curve

  • Apply appropriate curve-fitting models (four-parameter logistic or five-parameter logistic regression)

  • Assess curve quality through R² values (aim for >0.98) and residual analysis

• Sample Quantification:

  • Run samples in technical triplicates to assess assay variability

  • Include quality control samples of known concentration

  • Calculate concentration using only values falling within the linear range of the standard curve

• Data Normalization Options:

  • Normalize to total protein concentration of samples

  • Express as relative values to a control condition

  • Consider normalization to fresh weight or cell number for plant samples

• Statistical Tests:

  • For comparing two groups: t-test (paired or unpaired as appropriate)

  • For multiple groups: one-way or two-way ANOVA followed by appropriate post-hoc tests

  • For non-normally distributed data: non-parametric alternatives (Mann-Whitney, Kruskal-Wallis)

• Advanced Statistical Considerations:

  • Account for batch effects if samples were processed on different plates/days

  • Consider hierarchical/mixed models when analyzing nested experimental designs

  • Perform power analysis to ensure adequate sample size for detecting biologically meaningful differences

• Data Reporting:

  • Include measures of central tendency (mean/median) and dispersion (SD/SEM)

  • Report both raw and normalized data when possible

  • Clearly state significance thresholds and statistical tests used

How does the At5g38810 antibody performance compare to antibodies against related proteins?

When evaluating the At5g38810 antibody in comparison to antibodies against related proteins, several factors should be considered:

• Specificity Comparison: The At5g38810 antibody is polyclonal and raised against recombinant full-length protein , which potentially provides recognition of multiple epitopes. This differs from antibodies designed against specific peptide regions, which may offer higher specificity but potentially lower sensitivity. Researchers should consider testing for cross-reactivity with closely related Arabidopsis proteins (e.g., At5g38790, At5g38800, At5g38910) that may share sequence similarity.

• Application Versatility: While the At5g38810 antibody is validated for ELISA and Western blot applications , antibodies against related proteins may be validated for additional techniques such as immunoprecipitation, immunohistochemistry, or flow cytometry. The methodological requirements of your research should guide antibody selection.

• Performance Metrics: Systematically compare signal-to-noise ratio, detection limit, and batch-to-batch consistency across different antibodies. For quantitative applications, construct standard curves using recombinant proteins to compare dynamic range and sensitivity.

• Expression Pattern Analysis: Use comparative immunoblotting to evaluate whether At5g38810 and related proteins exhibit overlapping or distinct expression patterns across tissues and conditions, which may provide functional insights.

When designing experiments requiring detection of multiple related proteins, consider the species of origin for each antibody to enable simultaneous use without cross-reactivity of secondary antibodies.

What are the considerations for using the At5g38810 antibody in non-model plant species?

Applying the At5g38810 antibody in non-model plant species requires careful consideration of several factors:

These considerations help ensure that data generated using the At5g38810 antibody in non-model species is reliable and correctly interpreted.