At2g35615 Antibody

What is At2g35615 antibody and what is its target?

At2g35615 antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At2g35615 protein. The target protein corresponds to UniProt accession number Q3EBM5. This antibody specifically reacts with Arabidopsis thaliana and has been affinity purified to enhance specificity . The antibody serves as a valuable tool for detecting and studying the At2g35615 protein in plant molecular biology research.

What are the recommended applications for At2g35615 antibody?

At2g35615 antibody has been tested and validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications . These techniques allow researchers to detect and quantify the target protein in complex biological samples. When designing experiments using this antibody, researchers should implement proper controls to ensure specificity and account for potential cross-reactivity, as antibody validation is critical for reproducible research outcomes .

How should At2g35615 antibody be stored and handled?

Proper storage and handling are essential for maintaining antibody functionality. At2g35615 antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided as they can compromise antibody activity and specificity. The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . These storage conditions help maintain antibody stability and functionality over time.

What controls should be included when using At2g35615 antibody?

Implementing these controls is essential as research has shown that many commercially available antibodies lack proper validation, potentially leading to unreliable results . Antibody characterization should document that the antibody binds to the target protein, functions in complex protein mixtures, doesn't bind to unintended targets, and performs as expected under specific experimental conditions .

How can I validate the specificity of At2g35615 antibody for my experiments?

Validating antibody specificity is crucial before proceeding with experiments. For At2g35615 antibody, consider implementing the following validation approaches:

• Genetic validation: Use knockout or knockdown Arabidopsis lines lacking At2g35615 as negative controls.

• Orthogonal validation: Compare results with alternative methods of protein detection.

• Independent antibody validation: Test multiple antibodies against different epitopes of At2g35615.

• Signal validation: Confirm that signal intensity correlates with expected expression patterns.

• Western blot analysis: Verify a single band of appropriate molecular weight.

These validation steps are critical as research has shown that inadequate antibody characterization can cast doubt on experimental results . Documentation of these validation steps should be included in research publications to enhance reproducibility.

What are the optimal experimental design considerations for Western blotting with At2g35615 antibody?

When designing Western blot experiments with At2g35615 antibody, consider the following methodological approach:

• Sample preparation: Extract total protein from Arabidopsis tissues using a buffer containing protease inhibitors.

• Protein denaturation: Heat samples at 95°C for 5 minutes in sample buffer containing SDS and reducing agent.

• Gel electrophoresis: Separate proteins using 10-12% SDS-PAGE gels.

• Transfer conditions: Use PVDF membrane with standard transfer buffer (25mM Tris, 192mM glycine, 20% methanol).

• Blocking: Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute At2g35615 antibody (optimal dilution determined experimentally, typically 1:1000) in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG at 1:5000 dilution.

• Detection: Visualize using enhanced chemiluminescence.

How should I design ELISA experiments using At2g35615 antibody?

For ELISA experiments with At2g35615 antibody, implement the following methodological approach:

• Plate preparation: Coat 96-well plates with capture antibody or antigen.

• Blocking: Block plates with appropriate buffer (e.g., 1-3% BSA in PBS) to prevent non-specific binding.

• Sample preparation: Prepare protein extracts from Arabidopsis tissues with consistent protein concentration.

• Primary antibody: Apply At2g35615 antibody at optimized dilution.

• Detection system: Use appropriate HRP-conjugated secondary antibody and substrate.

• Data analysis: Generate standard curves and analyze samples in technical and biological replicates.

This experimental design should include appropriate normalization procedures to eliminate systematic bias and statistical analyses to assess differential expression . Two-color detection systems may be employed to compare expression levels between different samples, similar to methods developed for antibody microarrays.

How can I address non-specific binding issues with At2g35615 antibody?

Non-specific binding is a common challenge with antibodies. If experiencing high background or multiple bands with At2g35615 antibody, implement these methodological strategies:

• Optimize blocking conditions: Test different blocking agents (BSA, non-fat dry milk, casein) and concentrations.

• Increase washing stringency: Use higher salt concentration or add detergents like Tween-20 to washing buffers.

• Adjust antibody concentration: Titrate the antibody to find optimal concentration that maximizes specific signal while minimizing background.

• Pre-absorb antibody: Incubate with proteins from negative control samples to remove cross-reactive antibodies.

• Optimize incubation conditions: Adjust temperature, time, and buffer composition.

These approaches are particularly important given that many commercially available antibodies have been found to be non-specific, as documented with AT2 receptor antibodies in cardiovascular research . Thorough validation is essential to ensure signal specificity.

What methodological approaches can help resolve contradictory results when using At2g35615 antibody?

When faced with contradictory results using At2g35615 antibody, implement these systematic troubleshooting steps:

• Validate antibody lot-to-lot consistency: Test multiple lots of the antibody to identify potential variability.

• Evaluate epitope accessibility: Different sample preparation methods may affect epitope exposure.

• Consider post-translational modifications: These can affect antibody binding and lead to inconsistent results.

• Assess technical variability: Systematically evaluate each step of your protocol to identify sources of variability.

• Implement orthogonal approaches: Use alternative methods (e.g., mass spectrometry) to confirm protein identity and abundance.

This systematic approach is important because antibody performance can vary significantly between experimental conditions, affecting reproducibility. Research has shown that antibody characterization is critical to enhance reproducibility in biomedical research .

How can I adapt At2g35615 antibody protocols for different plant tissues or growth conditions?

Adapting protocols for different tissues or conditions requires methodological adjustments:

• Extraction buffer optimization: Different tissues may require specific buffer compositions to efficiently extract proteins while preserving antibody epitopes.

• Sample preparation modifications:

  • Woody tissues: Include additional grinding steps and stronger extraction buffers

  • High-lipid tissues: Add additional detergents or organic solvent treatments

  • Stress-treated samples: Account for increased secondary metabolites that may interfere with antibody binding

• Detection system sensitivity adjustment: Different tissues may have varying expression levels requiring adjusted exposure times or more sensitive detection systems.

• Background reduction strategies: Tissue-specific compounds may contribute to background; implement specific pre-treatments to reduce interference.

These adaptations must be validated for each new experimental condition, as antibody performance can vary significantly across different sample types .

What methodological approaches can be used to study protein-protein interactions involving the At2g35615 protein?

To study protein-protein interactions involving At2g35615, researchers can employ these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse Arabidopsis cells in non-denaturing buffer

  • Incubate lysate with At2g35615 antibody and protein A/G beads

  • Wash and elute complexes

  • Analyze interacting partners by mass spectrometry or Western blotting

• Proximity ligation assay (PLA):

  • Fix and permeabilize plant cells

  • Incubate with At2g35615 antibody and antibody against potential interactor

  • Apply oligonucleotide-conjugated secondary antibodies

  • Perform rolling circle amplification if antibodies are in proximity

  • Visualize interaction through fluorescence microscopy

• Bimolecular fluorescence complementation (BiFC):

  • Clone At2g35615 and potential interacting proteins fused to complementary fragments of fluorescent protein

  • Express in plant cells

  • Analyze reconstituted fluorescence by microscopy

These approaches require thorough controls to ensure specificity, including the use of unrelated antibodies and knockout lines .

How can I quantitatively assess At2g35615 protein expression levels across different experimental conditions?

For quantitative assessment of At2g35615 across experimental conditions, implement these methodological approaches:

• Quantitative Western blotting:

  • Include standard curve with recombinant protein

  • Use fluorescent secondary antibodies for wider linear range

  • Analyze band intensity with appropriate software

  • Normalize to loading controls (e.g., actin, tubulin)

• Quantitative ELISA:

  • Develop standard curve using purified recombinant protein

  • Process samples in technical triplicates

  • Include appropriate normalization controls

• Statistical analysis:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests (e.g., t-test, ANOVA)

  • Consider power analysis to determine sample size requirements

Normalization procedures are essential to eliminate systematic bias, and appropriate statistical analyses are crucial to assess differential expression, following methodologies developed for antibody microarrays .

What are the considerations for multiplexing At2g35615 antibody with other antibodies for co-localization studies?

When multiplexing At2g35615 antibody with other antibodies, consider these methodological approaches:

• Antibody compatibility assessment:

  • Ensure primary antibodies are raised in different host species

  • Verify no cross-reactivity between secondary antibodies

  • Test each antibody individually before multiplexing

• Sequential immunostaining protocol:

  • First round: Apply At2g35615 antibody, detect and image

  • Stripping step: Remove antibodies while preserving sample integrity

  • Second round: Apply second primary antibody and detect

• Controls for multiplexed staining:

  • Single antibody controls

  • Secondary antibody-only controls

  • Absorption controls with blocking peptides

• Analysis of co-localization:

  • Use appropriate software for quantitative co-localization analysis

  • Apply statistical methods (e.g., Pearson's correlation coefficient)

  • Include non-colocalized controls for threshold setting

These approaches help ensure that observed co-localization is not an artifact of cross-reactivity or bleed-through between fluorescence channels .