At2g29810 Antibody

What is the At2g29810 gene and what protein does it encode?

At2g29810 is a gene locus on chromosome 2 of Arabidopsis thaliana that encodes a protein identified by UniProt accession number O82375. The gene is expressed in various tissues throughout the plant's development. When designing experiments targeting this protein, researchers should consider its expression patterns across different developmental stages and in response to various environmental conditions .

How can I validate the specificity of At2g29810 antibodies?

Validating antibody specificity is critical given the widespread problems with commercial antibodies. Recommended validation approaches include:

• Western blot analysis comparing wild-type and knockout/knockdown plants

• Peptide competition assays by pre-incubating the antibody with the immunizing peptide

• Immunoprecipitation followed by mass spectrometry

• Correlation of protein detection with transcript levels from RT-PCR

• Reactivity testing against recombinant At2g29810 protein

Studies have shown that commercial antibodies can produce nonspecific signals, making validation essential before experimental use . All validation experiments should include appropriate negative controls, particularly tissue from knockout plants.

What are the storage and handling recommendations for At2g29810 antibodies?

For optimal antibody performance:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• The standard formulation contains 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as preservative

• For working solutions, store at 4°C for no longer than one week

• Monitor expiration dates and storage conditions carefully

Improper storage can lead to antibody degradation, resulting in decreased specificity and sensitivity.

What applications have been validated for At2g29810 antibodies?

According to manufacturer specifications, At2g29810 antibodies have been validated for:

• Enzyme-linked immunosorbent assay (ELISA)

• Western blotting (WB)

Other applications require validation by individual researchers before use in critical experiments .

How should I design a Western blot experiment using At2g29810 antibody?

A methodologically sound Western blot protocol includes:

• Sample preparation: Harvest plant tissue, flash-freeze in liquid nitrogen, and grind into a fine powder. Extract proteins using a buffer containing detergent, protease inhibitors, and reducing agents.

• Protein separation: Load 10-15 μg total protein per lane on SDS-PAGE gels.

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane.

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

• Primary antibody incubation: Dilute At2g29810 antibody (starting at 1:1000) in blocking buffer and incubate overnight at 4°C.

• Washing: Wash membrane 3× with TBST, 10 minutes each.

• Secondary antibody incubation: Incubate with HRP-conjugated anti-rabbit IgG (1:5000) for 1 hour.

• Detection: Develop using chemiluminescence and image.

Include positive controls (wild-type tissue), negative controls (knockout tissue if available), and loading controls (anti-actin or anti-tubulin) .

Can At2g29810 antibodies be used for chromatin immunoprecipitation (ChIP) studies?

While not explicitly validated for ChIP, researchers can adapt At2g29810 antibodies for chromatin studies with careful optimization:

• The antibody must recognize native (non-denatured) protein conformations

• Crosslinking and chromatin extraction protocols need optimization for plant tissues

• Include appropriate controls: IgG control, input DNA, and ideally a knockout control

• Validate enrichment using qPCR of known targets before proceeding to sequencing

Recent work with plant chromatin proteins demonstrates the potential of custom antibodies for ChIP applications in studying transcriptional regulation in Arabidopsis .

How do I determine the optimal antibody concentration for my experiment?

To determine optimal antibody concentration:

• Perform a titration experiment using serial dilutions (1:500, 1:1000, 1:2000, 1:5000)

• Test each dilution under identical conditions

• Select the concentration that provides the best signal-to-noise ratio

• Document optimal conditions for reproducibility

Optimal concentrations typically differ between applications (Western blot vs. ELISA vs. immunohistochemistry).

What is the recommended sample preparation protocol for plant tissues?

Effective sample preparation for Arabidopsis tissues includes:

• Harvest tissue and immediately freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining freezing conditions

• Extract in buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

  • 1 mM DTT or 5 mM β-mercaptoethanol

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect supernatant and quantify protein concentration

• Prepare samples in SDS-PAGE loading buffer

For membrane proteins or nuclear proteins, specialized extraction protocols may be necessary.

How can I troubleshoot high background or non-specific binding?

To minimize non-specific binding:

• Increase blocking time (from 1 hour to overnight)

• Use alternative blocking agents (5% BSA instead of milk)

• Add 0.1-0.5% Tween-20 to wash and antibody incubation buffers

• Pre-absorb the antibody with proteins from non-target species

• Optimize antibody dilution (excessive concentration increases background)

• Ensure thorough washing between steps (minimum 3× 10 minutes)

Non-specific binding is particularly common with polyclonal antibodies and requires systematic troubleshooting.

How can I use At2g29810 antibody to study protein interactions?

For protein interaction studies:

• Co-immunoprecipitation (Co-IP):

  • Use At2g29810 antibody to pull down the protein complex

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Include stringent controls (IgG control, knockout tissue)

• Proximity ligation assay (PLA):

  • Combine At2g29810 antibody with antibodies against potential interacting partners

  • Visualize interactions in situ through fluorescent detection

• Immunoaffinity purification:

  • Immobilize At2g29810 antibody on a solid support

  • Isolate protein complexes from plant extracts

  • Identify components by mass spectrometry

This approach has been successfully used to study protein complexes in plants, including chromatin remodeling complexes .

How can I design experiments to study At2g29810 protein dynamics during environmental stress responses?

To study protein dynamics during stress responses:

• Design time-course experiments with appropriate stress treatments

• Include multiple biological replicates (minimum n=3)

• Harvest tissues at defined time points

• Process all samples simultaneously using standardized protocols

• Quantify protein levels using:

  • Quantitative Western blotting with fluorescent secondary antibodies

  • ELISA for higher throughput

• Normalize to appropriate housekeeping proteins

• Correlate protein changes with transcript dynamics using RT-qPCR

• Apply statistical analyses to determine significance

Studies examining protein responses to plant hormones like ABA have successfully utilized similar approaches to track dynamic protein changes .

How do I interpret contradictory results between antibody-based detection and transcript expression data?

When protein and transcript data don't align:

• Consider post-transcriptional regulation mechanisms:

  • microRNA-mediated silencing

  • Alternative splicing

  • Translation efficiency differences

• Evaluate protein stability and turnover:

  • Some proteins have long half-lives despite low transcript levels

  • Others undergo rapid degradation despite high transcript abundance

• Examine experimental limitations:

  • Antibody accessibility to the epitope

  • Protein extraction efficiency

  • Post-translational modifications affecting antibody binding

• Perform additional experiments:

  • Pulse-chase labeling to measure protein turnover

  • Proteasome inhibitor treatment to assess degradation pathways

  • Analysis of polysome-bound transcripts to measure translation

Protein levels frequently don't correlate directly with transcript levels due to the complexity of gene expression regulation.

What controls should I include when using At2g29810 antibody?

A comprehensive control strategy includes:

Proper controls are essential for accurate interpretation, especially given the documented issues with antibody specificity in research settings .

Can At2g29810 antibody be used across different Arabidopsis ecotypes or related plant species?

For cross-ecotype or cross-species applications:

• Analyze protein sequence conservation in the epitope region

• Conduct preliminary Western blots to assess cross-reactivity

• Consider epitope conservation when interpreting results:

  • High conservation suggests potential cross-reactivity

  • Variations may affect antibody binding affinity

• Include appropriate positive controls from Arabidopsis thaliana Col-0

• Validate any novel cross-reactivity with additional methods

Even single amino acid changes in epitope regions can significantly impact antibody recognition.

How can I use At2g29810 antibody to study post-translational modifications?

To investigate post-translational modifications:

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Identify modified forms by shifts in migration pattern

• Phosphorylation analysis:

  • Treat samples with phosphatase before Western blotting

  • Compare migration patterns before and after treatment

  • Use phosphorylation-specific detection methods as complementary approaches

• IP-mass spectrometry:

  • Immunoprecipitate At2g29810 protein

  • Analyze by mass spectrometry to identify modifications

  • Compare modification patterns under different conditions

Similar approaches have been used to study modifications of plant chromatin proteins in response to environmental stimuli .

What are the relative advantages of different detection methods using At2g29810 antibody?

Comparison of detection methodologies:

Method selection should align with specific research questions and available resources.

How does antibody affinity affect experimental outcomes in different applications?

Antibody affinity impacts:

• Sensitivity: Higher affinity antibodies detect lower protein concentrations

• Specificity: Appropriate affinity balances detection of target vs. cross-reactivity

• Washing stringency: Higher affinity antibodies tolerate more stringent washing

• Immunoprecipitation efficiency: Directly proportional to antibody affinity

• Required concentration: Inversely proportional to affinity

For quantitative applications, consistent antibody lots with characterized affinity are essential for reproducible results.

Understanding these considerations will enable researchers to design robust experiments using At2g29810 antibodies for studying plant molecular biology and developmental processes in Arabidopsis thaliana.

What statistical approaches are recommended for analyzing quantitative Western blot data using At2g29810 antibody?

For rigorous quantitative analysis:

• Include at least three biological replicates per condition

• Normalize band intensity to loading controls

• Use appropriate statistical tests:

  • t-test for two-group comparisons

  • ANOVA followed by post-hoc tests for multiple groups

  • Non-parametric alternatives if normality assumptions are violated

• Report both effect size and p-values

• Consider using specialized software for densitometry analysis

• Validate important findings with complementary methods

How can I integrate antibody-based protein detection with other omics approaches?

Multi-omics integration strategies:

• Correlate protein levels (detected by At2g29810 antibody) with:

  • Transcriptomics data from RNA-Seq

  • Epigenomic data from ChIP-Seq

  • Metabolomic profiles related to the protein's function

• Use antibody-based approaches to validate key findings from high-throughput studies

• Apply computational approaches to integrate multiple data types:

  • Network analysis

  • Pathway enrichment

  • Multi-omics correlation analysis

Similar integrative approaches have been successfully applied to study protein function in plant stress responses .