At1g33530 Antibody

What is the At1g33530 protein in Arabidopsis thaliana and what cellular functions does it serve?

At1g33530 is a protein encoded in the Arabidopsis thaliana genome, specifically located on chromosome 1. Based on genomic annotation, this protein is involved in cellular processes that require further characterization through experimental approaches. The protein has been assigned UniProt accession number Q9C800 , indicating it has been documented in protein databases, though its precise functional characterization remains an active area of research. Studying this protein typically involves both genomic approaches (like PCR assays) and protein-level analysis with antibodies designed specifically against the At1g33530 protein.

What validation methods should be employed to confirm At1g33530 Antibody specificity?

Antibody validation requires multiple complementary approaches:

• Western blot analysis: Confirm a single band of the expected molecular weight is detected in wild-type Arabidopsis samples, with reduced or absent signal in knockout mutants

• Immunoprecipitation followed by mass spectrometry: Verify the antibody pulls down primarily the target protein

• Cross-reactivity testing: Compare detection patterns between different plant species or tissues with known expression patterns

• Application-specific validation: For ELISA applications, establish standard curves using recombinant protein

For polyclonal antibodies like the At1g33530 antibody, batch-to-batch variation necessitates validation of each lot. The antibody is specifically designed to recognize the recombinant Arabidopsis thaliana At1g33530 protein (its immunogen) , making verification with this recombinant protein particularly valuable for validation studies.

What are the recommended storage and handling conditions for maximizing At1g33530 Antibody stability?

For optimal stability and performance of the At1g33530 Antibody:

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

• Avoid repeated freeze-thaw cycles

• For working solutions, store in small aliquots to minimize freeze-thaw damage

• The antibody is preserved in 0.03% Proclin 300 and supplied in 50% Glycerol, 0.01M PBS at pH 7.4

• When handling, use sterile technique and maintain cold chain

• For long-term storage, -80°C is preferred to maintain binding capacity

• Prior to experiments, equilibrate to room temperature gradually before opening the tube to prevent condensation

Maintaining proper storage conditions is critical as antibody degradation can lead to reduced specificity, increased background, and diminished signal intensity in experimental applications.

How should Western blot protocols be optimized when using At1g33530 Antibody?

For optimal Western blot results with At1g33530 Antibody:

What considerations are important when using At1g33530 Antibody for immunolocalization studies?

For successful immunolocalization with At1g33530 Antibody:

• Fixation optimization: Test multiple fixatives (4% paraformaldehyde, glutaraldehyde combinations) to preserve epitope accessibility while maintaining cellular structure

• Antigen retrieval: Plant tissues often require citrate buffer or enzymatic antigen retrieval methods

• Permeabilization: Critical for plant tissues with cell walls; use detergents like Triton X-100 (0.1-0.5%) or digestive enzymes

• Blocking: Extended blocking (2+ hours) with 3-5% BSA or normal serum from the secondary antibody species

• Antibody dilution: Start with 1:100 to 1:500, with overnight incubation at 4°C

• Controls: Include negative controls (secondary antibody only, pre-immune serum) and positive controls (tissues with known expression)

• Signal amplification: Consider tyramide signal amplification if the protein is low abundance

Because the At1g33530 Antibody has been validated for ELISA and Western blot applications , optimization for immunolocalization may require additional protocol adjustments. The polyclonal nature of this antibody provides multiple epitope recognition which can be advantageous for detecting native protein in fixed tissues.

How can At1g33530 Antibody be integrated with gene expression studies using RT-qPCR?

Integration of antibody detection with gene expression analysis creates powerful multi-level experimental designs:

• Correlation analysis:

  • Use PrimePCR Probe Assay for At1g33530 gene expression measurement

  • Quantify protein levels by Western blot or ELISA using the At1g33530 Antibody

  • Analyze protein-to-mRNA ratios across developmental stages or treatments

• Experimental workflow:

  • Split samples for parallel processing:

    • RNA extraction for RT-qPCR using AT1G33530-specific primers

    • Protein extraction for antibody-based detection

  • Compare expression patterns and calculate correlation coefficients

• Validation strategy:

  • Confirm knockdown/overexpression at both transcript level (RT-qPCR) and protein level (antibody detection)

  • Use for transgenic line validation

• Temporal studies:

  • Track changes in mRNA vs. protein levels during developmental stages or stress responses

  • Identify post-transcriptional regulation by detecting discrepancies between transcript and protein levels

This integrated approach combines the amplicon context sequence information from PrimePCR assays with protein detection capabilities of the antibody for comprehensive gene function analysis.

What approaches can be used to determine binding kinetics and affinity of At1g33530 Antibody to its target antigen?

For detailed binding characterization of At1g33530 Antibody:

• Surface Plasmon Resonance (SPR):

  • Immobilize recombinant At1g33530 protein on sensor chip

  • Flow antibody at various concentrations

  • Measure association (ka) and dissociation (kd) rate constants

  • Calculate equilibrium dissociation constant (KD = kd/ka)

• Bio-Layer Interferometry (BLI):

  • Alternative to SPR with similar principles

  • Allows real-time measurement without microfluidics

• Isothermal Titration Calorimetry (ITC):

  • Measures heat changes during binding

  • Provides thermodynamic parameters (ΔH, ΔS, ΔG)

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Serial dilutions of antibody against fixed antigen concentration

  • Plot binding curve and calculate EC50

  • Less precise than SPR but accessible to most laboratories

For polyclonal antibodies like At1g33530 Antibody, these measurements represent average affinities across multiple epitope-specific antibody populations. The antigen affinity purification method used for this antibody should produce a preparation with good specific binding characteristics, though heterogeneity is expected compared to monoclonal antibodies.

How can computational modeling assist in understanding At1g33530 Antibody epitope recognition patterns?

Computational approaches provide valuable insights into antibody-antigen interactions:

• Epitope prediction:

  • B-cell epitope prediction algorithms can identify likely binding regions on At1g33530

  • Tools like BepiPred, Ellipro, or ABCpred analyze protein sequence for hydrophilicity, accessibility, and flexibility

• Structural modeling:

  • Generate 3D models of At1g33530 protein using homology modeling or AlphaFold2

  • Dock antibody variable regions to predicted epitopes

  • Energy minimization to refine interaction interfaces

• Binding mode analysis:

  • Similar to approaches described in search result , multiple binding modes can be identified

  • Each mode represents a distinct interaction between antibody paratopes and At1g33530 epitopes

  • Parameterization through shallow dense neural networks helps predict binding probabilities

• Cross-reactivity prediction:

  • Compare At1g33530 with homologous proteins from other species

  • Identify conserved vs. unique epitopes to predict potential cross-reactivity

  • Guide experimental validation of antibody specificity

These computational approaches complement experimental characterization and can help researchers understand the molecular basis of antibody recognition, potentially explaining experimental observations of specificity or cross-reactivity.

What strategies can overcome common challenges when using At1g33530 Antibody for chromatin immunoprecipitation (ChIP)?

While At1g33530 Antibody was primarily validated for ELISA and Western blot applications , adapting it for ChIP requires careful optimization:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.75-1.5%)

  • Vary crosslinking times (10-20 minutes)

  • Consider dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde for improved protein-DNA fixation

• Chromatin fragmentation:

  • Optimize sonication parameters for 200-500bp fragments

  • Monitor fragmentation by agarose gel electrophoresis

  • Consider enzymatic fragmentation alternatives

• Antibody binding conditions:

  • Increase antibody concentration (2-5x Western blot conditions)

  • Extended incubation (overnight at 4°C with rotation)

  • Add BSA (0.1-0.5%) to reduce non-specific binding

• Controls and validation:

  • Include IgG negative control

  • Use known target genes for positive control

  • Validate with sequential ChIP or ChIP-reChIP if appropriate

  • Validate enrichment by qPCR before sequencing

• Signal enhancement:

  • Consider antibody pooling from multiple lots

  • Implement carrier ChIP for low abundance targets

Since the At1g33530 Antibody is polyclonal and affinity-purified , it may recognize multiple epitopes on the target protein, potentially increasing ChIP efficiency if the protein maintains these epitopes in its native chromatin context.

How should researchers address inconsistent results between different lots of At1g33530 Antibody?

Polyclonal antibody variation between lots is a common challenge requiring systematic approaches:

• Lot-to-lot comparison protocol:

  • Run parallel Western blots with identical samples

  • Construct standard curves with recombinant protein

  • Document optimal dilutions and incubation conditions for each lot

• Performance standardization:

  • Maintain reference samples with known reactivity

  • Normalize data to these standards when changing lots

  • Consider pooling antibodies from multiple lots for critical experiments

• Validation parameters:

  Parameter	Acceptance Criteria	Method
  Specificity	Single band of expected size	Western blot
  Sensitivity	Detection limit ≤ 50ng target	Titration curve
  Background	Signal:noise ratio > 10:1	Western blot
  Cross-reactivity	Minimal binding to non-target proteins	Testing against knockout samples

• Documentation practices:

  • Record lot numbers in all experimental notes

  • Document all optimization parameters for each lot

  • Consider preparing large batches of working dilution to minimize variation in long-term projects

Since the At1g33530 Antibody is produced against a recombinant protein immunogen , requesting information about the exact immunogen sequence from the manufacturer can help understand potential epitope differences between lots.

What are the most effective approaches for reducing high background when using At1g33530 Antibody in immunodetection?

High background is a common issue that can be systematically addressed:

• Blocking optimization:

  • Test alternative blocking agents (milk, BSA, normal serum, commercial blockers)

  • Increase blocking time (2-16 hours)

  • Add 0.1-0.5% Tween-20 or Triton X-100 to blocking buffer

• Antibody conditions:

  • Further dilute primary antibody (try 1:2000 to 1:5000)

  • Reduce incubation temperature to 4°C

  • Add 0.1-0.5% detergent to antibody dilution buffer

  • Pre-absorb antibody with plant extract from knockout mutants

• Washing modifications:

  • Increase wash volume and duration

  • Add salt to wash buffer (up to 500mM NaCl)

  • Include 0.05-0.1% SDS in wash buffer for Western blots

• Detection system adjustments:

  • Reduce secondary antibody concentration

  • Shorter exposure times for chemiluminescence

  • Use fluorescent secondaries for better signal:noise ratio

• Sample preparation:

  • Include additional clarification steps during extraction

  • Pre-clear lysates with Protein A/G beads

  • Add protease inhibitors to prevent degradation products

For polyclonal antibodies like At1g33530 Antibody, some background is expected due to the heterogeneous antibody population. The antibody's antigen affinity purification helps reduce this, but optimization of the above parameters may still be necessary for different applications.

How can researchers appropriately cite and document At1g33530 Antibody use in scientific publications?

Proper documentation of antibody use is critical for reproducibility:

• Required citation information:

  • Full antibody name: Anti-At1g33530 Antibody

  • Manufacturer and catalog number: CSB-PA871683XA01DOA

  • Host species: Rabbit

  • Clonality: Polyclonal

  • Antigen: Recombinant Arabidopsis thaliana At1g33530 protein

  • RRID (Research Resource Identifier) if available

• Methods section details:

  • Lot number used

  • Dilution factors for each application

  • Incubation conditions (time, temperature)

  • Detection method

  • Blocking reagents

  • Quantification methodology

• Validation documentation:

  • Description of controls used

  • Reference to validation experiments (knockout controls, etc.)

  • Images of full blots/gels in supplementary materials

  • Quantification methods and normalization approach

• Data availability:

  • Raw images deposited in public repositories

  • Transparent reporting of all replicates

  • Documentation of any image processing

Following these guidelines aligns with best practices in antibody reporting and enhances experimental reproducibility across different laboratories studying At1g33530 and related proteins in Arabidopsis thaliana.

How can At1g33530 Antibody be incorporated into high-throughput automated immunoassay platforms?

Adapting the At1g33530 Antibody for automated platforms requires systematic optimization:

• Platform compatibility assessment:

  • Evaluate binding stability under high-throughput conditions

  • Test performance in miniaturized reaction volumes (5-20μL)

  • Assess buffer compatibility with automated systems

• Automation-specific optimizations:

  • Concentrate antibody stock to accommodate small volumes

  • Adjust incubation times for robotic handling

  • Develop calibration standards for quantitative analysis

• Multiplex integration strategies:

  • Label antibody with fluorophores/biotin for multiplex detection

  • Validate absence of cross-reactivity with other targets in multiplex panel

  • Establish detection thresholds in multiplex context

• Quality control measures:

  • Implement automated QC checkpoints

  • Develop control samples with known At1g33530 concentrations

  • Establish acceptance criteria for each run

High-throughput platforms could significantly enhance research productivity for projects requiring large-scale Analysis of At1g33530 expression across multiple conditions or genetic backgrounds, similar to the type of advanced analysis described for other antibodies in systems biology contexts .

What approaches can extend the utility of At1g33530 Antibody beyond Arabidopsis to other plant model systems?

Extending At1g33530 Antibody applications across species:

• Cross-reactivity analysis:

  • Perform sequence alignment of At1g33530 with homologs in target species

  • Identify conserved epitope regions

  • Test antibody against recombinant proteins or lysates from multiple species

• Optimization for diverse plant materials:

  Plant Material	Extraction Buffer Modifications	Protocol Adjustments
  Woody species	Add PVP (2-5%) and PVPP (1-2%)	Increased extraction time
  High-phenolic tissues	Add β-mercaptoethanol (5-10mM)	Additional clarification steps
  Recalcitrant species	Add SDS (0.1-0.5%)	Heat treatment during extraction

• Validation strategy:

  • Western blot with predicted molecular weight confirmation

  • Immunoprecipitation followed by mass spectrometry

  • Parallel analysis with species-specific genetic tools (RNAi, CRISPR)

• Heterologous expression systems:

  • Express At1g33530 orthologs in E. coli or yeast

  • Use for antibody validation and cross-reactivity assessment

  • Develop as positive controls for other species

This approach builds on methodologies similar to those employed in antibody specificity studies , where binding modes to related epitopes are characterized through experimental and computational approaches.

How might computational biology approaches improve interpretation of At1g33530 Antibody experimental results?

Advanced computational methods enhance antibody-based research:

• Machine learning for image analysis:

  • Train algorithms to quantify immunofluorescence patterns

  • Automated Western blot band quantification

  • Unbiased identification of subcellular localization patterns

• Systems biology integration:

  • Network analysis incorporating At1g33530 antibody-derived protein data

  • Integration with transcriptomics using corresponding PCR assays

  • Pathway enrichment analysis with protein interaction data

• Biophysical modeling approaches:

  • Similar to those described in search result , develop models of antibody-antigen interactions

  • Incorporate binding energy calculations

  • Predict effects of mutations or post-translational modifications

• Meta-analysis tools:

  • Cross-experiment normalization methods

  • Statistical approaches for integrating quantitative antibody data

  • Bayesian frameworks for interpreting variable results

These computational approaches transform raw antibody-generated data into biological insights, allowing researchers to place At1g33530 in broader functional contexts within plant biology.