At1g36730 Antibody

What is the At1g36730 protein and why is it studied in Arabidopsis research?

At1g36730 is a protein encoded by the At1g36730 gene in Arabidopsis thaliana, an important model organism in plant biology. While the specific function information is limited in the provided resources, researchers typically study such proteins to understand plant cellular functions, developmental processes, and stress responses. This protein may be involved in critical pathways that contribute to plant growth regulation, environmental adaptation, or other essential cellular processes. Studying it requires specific antibodies for detection and characterization in experimental systems .

What applications are validated for the At1g36730 antibody?

The At1g36730 antibody has been validated for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications. These techniques allow researchers to detect and quantify the presence of the target protein in various sample preparations. The antibody is specifically designed to ensure identification of the antigen in these experimental contexts. Additional applications may be possible but would require further validation by the researcher to confirm specificity and sensitivity in those experimental systems .

How should the At1g36730 antibody be stored to maintain optimal activity?

The At1g36730 antibody should be stored at -20°C or -80°C upon receipt. It's critical to avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce its effectiveness. The antibody is provided in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. Proper storage is essential for maintaining antibody performance across experiments and ensuring reproducible results in your research applications .

How do you optimize Western blot protocols for At1g36730 detection in plant samples?

Optimizing Western blot protocols for At1g36730 detection requires careful consideration of several parameters:

• Sample preparation: Extract proteins from Arabidopsis tissues using buffers containing protease inhibitors to prevent degradation. Consider comparing different extraction methods (e.g., mechanical disruption, chemical lysis) to determine optimal protein yield.

• Protein loading: Load 4-10 μg of total protein per well, based on protocols established for similar plant antibodies. This range allows for detection while minimizing background signal .

• Blocking conditions: Use 5% milk or BSA in TBS-T for 1 hour at room temperature to reduce non-specific binding .

• Antibody dilution: Start with a 1:5000 dilution for Western blot applications, adjusting as needed based on signal strength and background levels .

• Detection method: Both chemiluminescent and colorimetric detection systems are compatible, with exposure times typically ranging from 15 seconds to several minutes depending on protein abundance .

What controls should be included when using At1g36730 antibody for the first time?

When using the At1g36730 antibody for the first time, include the following controls to validate specificity and reliability:

• Positive control: Include samples known to express At1g36730 protein, such as wild-type Arabidopsis thaliana leaf extracts.

• Negative control: Use either:

  • Samples from knockout/knockdown lines lacking At1g36730 expression

  • Samples from tissues known not to express the target protein

  • Pre-immune serum or secondary antibody-only controls to assess non-specific binding

• Loading control: Include detection of a constitutively expressed protein (such as actin) to normalize for loading variations between samples. Actin antibodies like the monoclonal clone mAbGPa 10-B3 (AS16 3141) provide reliable loading controls in plant samples .

• Molecular weight verification: Include a molecular weight marker to confirm the detected band appears at the expected size.

How can At1g36730 antibody be used in combination with other antibodies for co-localization studies?

For co-localization studies combining At1g36730 antibody with other antibodies:

• Select compatible antibodies: Choose secondary antibodies with non-overlapping fluorophores if performing immunofluorescence. Ensure primary antibodies are raised in different host species (e.g., rabbit anti-At1g36730 with mouse anti-actin) to prevent cross-reactivity .

• Sequential probing protocol:

  • For Western blots: Strip and reprobe membranes between applications, or use differently colored detection systems

  • For immunohistochemistry: Apply primary antibodies sequentially with thorough washing steps, or simultaneously if they're from different host species

• Cross-reactivity testing: Prior to combined experiments, test each antibody individually to establish baseline signals and confirm no unexpected cross-reactivity occurs.

• Data integration: Collect and analyze signals from multiple channels, using appropriate controls to account for potential bleed-through or non-specific binding .

What are common causes of background noise when using At1g36730 antibody, and how can they be mitigated?

Common causes of background noise and their solutions include:

Optimizing each of these parameters systematically can significantly improve signal-to-noise ratio in your experiments .

How do you analyze and interpret inconsistent results when using At1g36730 antibody across different plant tissues?

When facing inconsistent results across different plant tissues:

• Evaluate protein expression levels: At1g36730 may be differentially expressed across tissues, leading to variable signal intensities. Consider using RT-PCR to correlate protein detection with mRNA expression.

• Assess extraction efficiency: Different plant tissues require optimized protein extraction protocols. Compare multiple extraction methods and buffers to ensure efficient protein recovery from recalcitrant tissues.

• Adjust detection parameters: More abundant proteins may require higher antibody dilutions (1:10000) while less abundant proteins might need more concentrated antibody solutions (1:1000).

• Consider post-translational modifications: The protein may undergo tissue-specific modifications affecting antibody recognition. Verify with additional antibodies targeting different epitopes when available.

• Validate with complementary techniques: Combine Western blotting with immunoprecipitation, mass spectrometry, or immunohistochemistry to build a more complete picture of protein expression patterns .

What statistical approaches are recommended for quantifying At1g36730 protein levels across experimental conditions?

For rigorous quantification of At1g36730 protein levels:

• Replication requirements: Include at least three biological replicates and two technical replicates per condition.

• Normalization strategies:

  • Normalize to total protein using stain-free technology or Ponceau staining

  • Normalize to housekeeping proteins (actin, tubulin) using antibodies such as AS16 3141 for actin

  • Consider multiple reference proteins for more accurate normalization

• Quantification methods:

  • Use densitometry software (ImageJ, Image Lab) to measure band intensities

  • Apply background subtraction consistently across all samples

  • Generate standard curves when absolute quantification is required

• Statistical analysis:

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

  • Use non-parametric tests when normality cannot be assumed

  • Report effect sizes alongside p-values for comprehensive interpretation

• Data presentation: Present normalized data with error bars representing standard deviation or standard error, clearly indicating sample size and statistical significance.

How can At1g36730 antibody be adapted for chromatin immunoprecipitation (ChIP) studies?

Adapting the At1g36730 antibody for ChIP applications requires:

• Cross-linking optimization: Test different formaldehyde concentrations (0.75-1.5%) and incubation times (10-20 minutes) to preserve protein-DNA interactions without overfixing.

• Antibody validation: Confirm the antibody can recognize its native epitope in fixed conditions through pilot immunoprecipitation experiments.

• Protocol adjustments:

  • Increase antibody concentration (typically 2-5 μg per immunoprecipitation)

  • Extend incubation times (overnight at 4°C with rotation)

  • Optimize washing stringency to balance specificity with yield

• Controls:

  • Include input controls (pre-immunoprecipitation chromatin)

  • Perform mock immunoprecipitations (no antibody or with pre-immune serum)

  • Use positive control antibodies (e.g., histone H3) in parallel experiments

• Data analysis: Apply appropriate normalization to input DNA and negative controls when quantifying enrichment by qPCR or sequencing .

What approaches enable investigation of At1g36730 protein-protein interactions in living plant cells?

To study At1g36730 protein-protein interactions in vivo:

• Co-immunoprecipitation (Co-IP): Use At1g36730 antibody to pull down the protein complex from plant extracts, followed by mass spectrometry or Western blotting with antibodies against suspected interaction partners.

• Proximity-dependent labeling: Fuse At1g36730 to enzymes like BioID or APEX2, which biotinylate nearby proteins upon activation, allowing identification of proximal proteins regardless of interaction strength.

• Förster Resonance Energy Transfer (FRET): Create fluorescent protein fusions to At1g36730 and potential partners to measure energy transfer as an indicator of protein proximity in living cells.

• Bimolecular Fluorescence Complementation (BiFC): Split a fluorescent protein between At1g36730 and candidate interactors; fluorescence is reconstituted only when proteins interact.

• Controls and validation:

  • Include non-interacting protein pairs as negative controls

  • Validate interactions through multiple independent methods

  • Confirm biological relevance through functional assays .

How can phosphorylation or other post-translational modifications of At1g36730 be detected using the available antibody?

To detect post-translational modifications (PTMs) of At1g36730:

• Modification-specific detection:

  • Perform immunoprecipitation with the At1g36730 antibody followed by Western blotting with modification-specific antibodies (anti-phospho, anti-ubiquitin, etc.)

  • Use Phos-tag™ PAGE to separate phosphorylated from non-phosphorylated forms before immunoblotting

• Mass spectrometry analysis:

  • Immunoprecipitate At1g36730 under native conditions

  • Digest the purified protein and analyze by LC-MS/MS

  • Search for mass shifts corresponding to specific modifications

• Induction experiments:

  • Compare modification patterns under different conditions known to induce PTMs (stress treatments, hormone applications)

  • Use phosphatase inhibitors during sample preparation to preserve phosphorylation states

  • Include appropriate controls (phosphatase-treated samples) to confirm specificity

• Two-dimensional gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Detect At1g36730 by Western blotting

  • Identify PTM-dependent shifts in protein migration patterns .

How does the specificity of the polyclonal At1g36730 antibody compare with monoclonal alternatives for plant protein research?

When comparing polyclonal At1g36730 antibody with monoclonal antibodies in plant research:

What methodological considerations are important when comparing At1g36730 expression data across different Arabidopsis ecotypes or mutant lines?

When comparing At1g36730 expression across Arabidopsis variants:

• Genetic background standardization:

  • Generate all mutant lines in the same ecotype background

  • Include appropriate wild-type controls for each genetic background

  • Consider backcrossing lines to standardize genetic backgrounds when necessary

• Growth condition normalization:

  • Standardize all environmental parameters (light intensity, photoperiod, temperature, humidity)

  • Grow all genotypes simultaneously under identical conditions

  • Sample at equivalent developmental stages rather than chronological age

• Technical considerations:

  • Process all samples using identical extraction protocols

  • Include internal loading controls (actin) for normalization

  • Run samples from different genotypes on the same gel/blot when possible

  • Repeat experiments across multiple independent biological replicates

• Data interpretation:

  • Consider natural variation in protein expression between ecotypes

  • Account for potential differences in antibody affinity due to protein sequence polymorphisms

  • Validate Western blot findings with transcript analysis when appropriate

How can researchers incorporate At1g36730 antibody in multi-omics experimental designs studying plant stress responses?

For integrating At1g36730 antibody-based detection into multi-omics experimental designs:

• Coordinated sampling strategy:

  • Collect parallel samples for proteomics, transcriptomics, and metabolomics from the same experimental units

  • Implement precise timing of sample collection across all data types

  • Include sufficient biological replicates (minimum n=3) for statistical power across all analyses

• Proteomics integration:

  • Use At1g36730 antibody in targeted protein quantification (Western blot)

  • Complement with untargeted proteomics approaches (MS-based)

  • Apply consistent normalization strategies across techniques

• Cross-platform data analysis:

  • Correlate protein abundance with transcript levels to identify post-transcriptional regulation

  • Map protein changes to metabolic pathways using metabolomics data

  • Implement computational approaches to integrate multi-dimensional datasets

• Validation experiments:

  • Design functional studies based on integrated omics insights

  • Use genetic approaches (mutants, overexpression lines) to confirm biological significance

  • Apply systems biology modeling to predict network-level effects

This integrative approach allows researchers to position At1g36730 within broader cellular networks and understand its contribution to stress response mechanisms from multiple analytical perspectives .