At1g18020 Antibody

What is At1g18020 Antibody and what protein does it target?

At1g18020 Antibody (catalog number CSB-PA320520XA01DOA) is a polyclonal antibody that specifically recognizes the protein encoded by the At1g18020 gene in Arabidopsis thaliana (Mouse-ear cress). This protein (UniProt accession P0DI09) plays important roles in plant cellular processes. The antibody is typically supplied in concentrations of 2ml/0.1ml or 10mg, depending on research requirements .

When designing experiments, researchers should note that this antibody has been validated for specific applications including immunoblotting, immunohistochemistry, and immunoprecipitation. For optimal results, always verify the application suitability in the manufacturer's documentation.

How should I validate At1g18020 Antibody before experimental use?

Proper validation is critical for ensuring experimental reproducibility. Follow these methodological steps:

• Titration: Always titrate the antibody under experimental conditions. At high concentrations, antibodies can bind to low-affinity targets after saturating intended epitopes, increasing background signal .

• Positive and negative controls: Use Arabidopsis thaliana wild-type tissue as a positive control and either At1g18020 knockout lines or non-plant tissues as negative controls.

• Specificity verification: Perform a western blot to confirm single-band detection at the expected molecular weight.

• Cross-reactivity assessment: Test against related plant species if your research involves comparative studies.

These validation steps help establish confidence in antibody performance before proceeding with critical experiments.

What are the recommended storage conditions for At1g18020 Antibody?

For optimal performance and longevity of At1g18020 Antibody:

• Store aliquots at -20°C for long-term preservation

• Avoid repeated freeze-thaw cycles (limit to <5 cycles)

• For working solutions, store at 4°C for up to two weeks

• Add preservatives like sodium azide (0.02%) for solutions stored at 4°C

• Maintain sterile conditions during handling

Following these storage protocols maximizes antibody shelf-life and ensures consistent experimental results over time.

How should I optimize At1g18020 Antibody concentration for immunoblotting?

Optimization requires systematic titration using the following methodology:

• Prepare a dilution series (typically 1:500 to 1:5000) of At1g18020 Antibody

• Run identical protein samples from Arabidopsis thaliana tissue

• Perform immunoblotting under identical conditions for all dilutions

• Evaluate signal-to-noise ratio across all concentrations

• Select the dilution that provides the strongest specific signal with minimal background

This methodical approach identifies the optimal antibody concentration where target saturation occurs without off-target binding. Titration should be carried out under the exact conditions that will be used for experimental work, with antibody concentration as the only variable .

What blocking reagents work best with At1g18020 Antibody?

Selecting appropriate blocking reagents significantly impacts specificity:

Begin with 5% non-fat milk in TBS-T, then test alternatives if background issues persist. For especially sensitive applications, consider using combination blocking strategies that incorporate both protein (BSA) and detergent (Tween-20) components.

How can I reduce background when using At1g18020 Antibody in immunohistochemistry?

To minimize background in plant tissue immunohistochemistry:

• Optimize fixation conditions (typically 4% paraformaldehyde)

• Increase washing duration and frequency (minimum 3×15 minutes)

• Include 0.1-0.3% Triton X-100 in blocking solutions

• Extend blocking duration to 2-3 hours at room temperature

• Dilute primary antibody appropriately based on titration

• Include 0.01% detergent in antibody incubation solutions

• Pre-absorb antibody with non-specific proteins if cross-reactivity persists

• Consider autofluorescence quenching steps for plant tissues

These methodical approaches address the specific challenges of plant tissue autofluorescence and protein complexity.

Why am I detecting multiple bands when using At1g18020 Antibody in western blotting?

Multiple bands may result from several research-relevant factors:

• Post-translational modifications: Phosphorylation, glycosylation, or ubiquitination can alter migration patterns

• Alternative splicing: At1g18020 may have splice variants

• Protein degradation: Incomplete protease inhibition during sample preparation

• Cross-reactivity: Antibody recognizing related epitopes in other proteins

• Sample preparation issues: Incomplete denaturation or reduction

Methodological solutions include:

• Verify sample integrity with fresh extraction and complete protease inhibitor cocktails

• Test different detergents for extraction (RIPA, NP-40, Triton X-100)

• Increase blocking stringency and washing steps

• Use gradient gels to better resolve proteins of similar molecular weights

• Consider immunoprecipitation followed by mass spectrometry to identify all detected proteins

How can I troubleshoot weak or absent signal when using At1g18020 Antibody?

When signal is weak or undetectable:

• Verify protein expression: Confirm At1g18020 expression in your experimental conditions using RT-PCR

• Check extraction efficiency: Different buffers may be required for membrane-associated proteins

• Optimize antibody concentration: Insufficient antibody reduces detection sensitivity

• Evaluate detection method sensitivity: Consider switching from colorimetric to chemiluminescent or fluorescent detection

• Assess epitope accessibility: Denaturation conditions may affect epitope structure

• Extend incubation time: Overnight primary antibody incubation at 4°C can improve signal

• Use signal enhancement: Tyramide signal amplification or biotin-streptavidin systems can boost sensitivity

These methodical approaches address various technical factors that may contribute to signal issues.

How can I use At1g18020 Antibody in co-immunoprecipitation studies?

For co-immunoprecipitation (Co-IP) to identify protein interaction partners:

• Buffer optimization: Use gentle lysis buffers containing 0.5-1% NP-40 or Digitonin to preserve protein-protein interactions

• Pre-clearing step: Incubate lysate with protein A/G beads before antibody addition to reduce non-specific binding

• Antibody immobilization: Consider cross-linking At1g18020 Antibody to beads using BS3 or DMP

• Control experiments: Include IgG controls and input sample lanes

• Elution conditions: Use gentle elution with peptide competition where possible

• Verification: Confirm interactions with reciprocal Co-IPs and proximity ligation assays

This advanced application can reveal important protein interaction networks involving the At1g18020 protein in plant cellular processes.

Can At1g18020 Antibody be used in chromatin immunoprecipitation (ChIP) experiments?

While not all antibodies are suitable for ChIP, methodological considerations for adapting At1g18020 Antibody include:

• Crosslinking optimization: Test both formaldehyde (protein-DNA) and DSG (protein-protein) crosslinking

• Sonication parameters: Optimize to generate 200-500bp DNA fragments

• Antibody validation: Verify specificity in plant nuclear extracts before ChIP

• Positive control regions: Include known binding regions if available

• Negative control regions: Include genomic regions not expected to bind

• Quantification method: Use both qPCR and sequencing for comprehensive analysis

For successful ChIP, validate antibody specificity for the native (non-denatured) conformation of At1g18020 protein, as western blot validation alone may not predict ChIP performance.

How can I apply antibody inverse folding models like IgDesign to optimize At1g18020 Antibody binding?

Recent advances in antibody engineering can enhance antibody performance through:

• Epitope mapping: Identify the precise binding site using peptide arrays or hydrogen-deuterium exchange mass spectrometry

• CDR optimization: Apply deep learning methods like IgDesign to redesign complementarity-determining regions (CDRs)

• Binding validation: Screen optimized antibodies using surface plasmon resonance (SPR)

• Affinity maturation: Generate variants with potentially improved binding characteristics

This advanced approach may be particularly valuable if current At1g18020 Antibody shows suboptimal performance in specific applications, allowing for custom antibody engineering to meet exact research requirements.

What considerations are important when using At1g18020 Antibody in flow cytometry with plant protoplasts?

Flow cytometry with plant protoplasts requires specific methodological adjustments:

• Protoplast preparation optimization: Use enzymatic digestion with carefully controlled concentrations and incubation times

• Fixation protocol: Optimize to preserve epitopes while maintaining cell integrity

• Permeabilization: Adjust conditions based on epitope location (surface vs. intracellular)

• Antibody concentration: Titrate specifically for flow cytometry conditions

• Controls: Include unstained, secondary-only, and isotype controls

• Autofluorescence compensation: Implement proper compensation strategies for plant cell autofluorescence

• Gating strategy: Develop specific strategies for plant protoplasts

These methodological considerations address the unique challenges of plant cell analysis by flow cytometry.

How can I quantify At1g18020 protein expression levels across different plant tissues or conditions?

For quantitative analysis across experimental conditions:

• Sample standardization: Normalize protein loading using total protein measurement methods like BCA

• Internal controls: Include constitutively expressed proteins (tubulin, actin) on each blot

• Standard curves: Generate a standard curve using recombinant protein if available

• Technical replicates: Run at least three technical replicates per biological sample

• Imaging methods: Use digital imaging with linear dynamic range

• Normalization strategy: Apply appropriate normalization to account for loading variations

• Statistical analysis: Apply appropriate statistical tests for comparisons between conditions

This quantitative approach enables robust comparative studies of At1g18020 protein expression across experimental conditions.