At5g10810 Antibody

What is the At5g10810 protein and why is it significant in plant biology research?

At5g10810 (UniProt: Q96319) is a protein-coding gene in Arabidopsis thaliana (Mouse-ear cress), a widely used model organism in plant molecular biology. This protein has gained significance in plant research due to its role in fundamental cellular processes. Antibodies targeting this protein allow researchers to investigate its expression patterns, localization, and functional relationships in various physiological and stress conditions. Unlike general protein studies, antibody-based approaches provide the advantage of detecting native protein in complex cellular environments without requiring protein purification or recombinant expression systems. When designing experiments with this antibody, researchers should consider tissue-specific expression patterns, developmental timing, and potential post-translational modifications that might affect epitope recognition .

How should researchers select appropriate positive and negative controls when working with At5g10810 Antibody?

Proper control selection is critical for antibody-based experiments. For positive controls, researchers should consider:

• Recombinant At5g10810 protein expression in heterologous systems

• Plant tissues with known high expression of At5g10810

• Tagged At5g10810 constructs in transgenic plants

For negative controls, consider:

• At5g10810 knockout/knockdown lines

• Tissues where the protein is not expressed

• Pre-immune serum controls

• Secondary antibody-only controls

The importance of proper validation cannot be overstated, as approximately 50% of commercial antibodies show some level of non-specific binding. This systematic approach helps distinguish specific signals from background, especially important when working with plant tissues that contain complex polysaccharides and secondary metabolites that can contribute to non-specific binding .

What are the key considerations for storing and handling At5g10810 Antibody to maintain its activity?

Proper antibody handling significantly impacts experimental reproducibility. Store At5g10810 Antibody according to manufacturer recommendations, typically at -20°C for long-term storage, with working aliquots kept at 4°C to minimize freeze-thaw cycles. When handling:

• Prepare small aliquots (10-50 μL) to prevent multiple freeze-thaw cycles

• Add carrier proteins (BSA 0.1-1%) for dilute solutions to prevent adsorption to tube walls

• Consider adding preservatives (0.02% sodium azide) for solutions stored at 4°C

• Document lot numbers and maintain validation data for each batch

Temperature fluctuations and improper storage can lead to antibody degradation and aggregation, resulting in reduced specificity and sensitivity. Studies have shown that antibody shelf life can be extended 2-3 fold with proper aliquoting and storage techniques. Some researchers report success with glycerol-based storage buffers (25-50% glycerol) for improved stability .

What are the optimal sample preparation protocols for At5g10810 detection in different Arabidopsis tissues?

Sample preparation varies significantly between different plant tissues and experimental techniques. For optimal At5g10810 detection:

For protein extraction:

• Young tissues: Grind in liquid nitrogen and extract with buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Mature/woody tissues: Require more stringent extraction:

  • Add 2% SDS to extraction buffer

  • Increase mechanical disruption time

  • Consider focused ultrasound for complete tissue disruption

For immunohistochemistry:

• Fixation in 4% paraformaldehyde (4-12 hours)

• Gradual dehydration through ethanol series

• Paraffin embedding and sectioning (5-10 μm)

• Antigen retrieval (critical step): 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 minutes

This methodological approach addresses the challenge of maintaining protein integrity while removing interfering compounds in plant tissues. Researchers should optimize protocols for specific tissue types, as extraction efficiency can vary by 30-40% between different plant organs .

How can researchers optimize Western blot conditions for At5g10810 Antibody to improve sensitivity and specificity?

Optimizing Western blot conditions for At5g10810 Antibody requires systematic adjustment of multiple parameters:

• Protein loading: Test 10-50 μg total protein per lane

• Blocking solutions:

  • Compare 5% non-fat dry milk vs. 3-5% BSA in TBS-T

  • Consider casein-based blockers for problematic samples

• Antibody dilution: Test range from 1:500 to 1:5000

• Incubation conditions:

  • Primary antibody: 4°C overnight vs. room temperature for 1-3 hours

  • Secondary antibody: Room temperature for 1 hour (1:5000-1:10000)

• Washing stringency: TBS-T (0.05% vs. 0.1% Tween-20)

• Detection methods: Compare chemiluminescence vs. fluorescence-based systems

Researchers should perform titration experiments to determine optimal antibody concentration, as both too high (leading to background) and too low (resulting in weak signals) concentrations compromise data quality. Evidence suggests that prolonged primary antibody incubation at 4°C often yields better signal-to-noise ratio compared to shorter room temperature incubations, with improvements of up to 40% in signal specificity .

What are effective strategies for immunoprecipitation using At5g10810 Antibody to study protein interactions?

For successful immunoprecipitation (IP) with At5g10810 Antibody:

• Pre-clearing strategy:

  • Incubate lysate with protein A/G beads (1 hour at 4°C) before adding antibody

  • Remove non-specific binding proteins by gentle centrifugation (1000 × g, 5 min)

• Antibody coupling:

  • Direct method: Covalently couple antibody to activated beads

  • Indirect method: Capture antibody using protein A/G beads

  • Use 2-5 μg antibody per 500 μg protein lysate

• Crosslinking considerations:

  • For transient interactions: Use formaldehyde (0.1-1%) or DSP (dithiobis-succinimidyl propionate)

  • Optimize crosslinking time (typically 10-30 minutes)

• Washing conditions:

  • Start with mild washing (TBS with 0.1% NP-40)

  • Increase stringency gradually (up to 500 mM NaCl)

  • Minimum 4-5 washes required

• Elution strategies:

  • Denaturing: SDS sample buffer at 95°C

  • Non-denaturing: Competitive elution with peptide

This methodological approach preserves native protein complexes while minimizing false positives. Studies show that pre-clearing can reduce non-specific background by 40-60%, and crosslinking can enhance detection of weak interactors by 30-50% .

How can researchers address non-specific binding issues with At5g10810 Antibody?

Non-specific binding is a common challenge when working with plant antibodies. To address this issue:

• Increase blocking stringency:

  • Test different blocking agents (milk, BSA, fish gelatin)

  • Increase blocking time (2-3 hours at room temperature)

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Titrate antibody concentration:

  • Perform systematic dilution series

  • Balance signal strength against background

• Add competing agents:

  • 0.1-0.5% non-ionic detergents

  • 100-500 mM NaCl to reduce ionic interactions

  • 1-5% polyethylene glycol to reduce hydrophobic interactions

• Absorption controls:

  • Pre-incubate antibody with recombinant target protein

  • Compare signal with and without absorption

• Sequential epitope exposure technique:

  • Gradually expose epitopes through controlled protein denaturation

  • Monitor signal-to-noise ratio at each step

Non-specific binding often occurs due to antibody cross-reactivity with structurally similar proteins. Research shows that approximately 30-50% of antibody-related experimental issues stem from non-specific binding. Systematic validation using knockout/knockdown controls is essential for confirming signal specificity .

What approaches can resolve discrepancies between At5g10810 protein levels detected by antibody versus transcript levels measured by RT-PCR?

Discrepancies between protein and mRNA levels are common and biologically significant. To investigate:

• Validate both detection methods independently:

  • Confirm antibody specificity using knockout/knockdown lines

  • Verify primer specificity for RT-PCR with sequencing

• Consider post-transcriptional regulation:

  • Analyze miRNA targeting the At5g10810 transcript

  • Examine RNA-binding protein interactions

  • Assess mRNA half-life through actinomycin D treatment

• Investigate post-translational regulation:

  • Analyze protein stability with cycloheximide chase assays

  • Examine ubiquitination status through IP followed by ubiquitin blotting

  • Assess proteasome involvement using MG132 treatment

• Time-course experiments:

  • Compare protein and mRNA kinetics following stimulus

  • Identify temporal delays between transcription and translation

• Quantification methods:

  • Use absolute quantification for both protein (with recombinant standards) and mRNA

  • Apply appropriate normalization strategies

Research demonstrates that protein and corresponding mRNA levels correlate only moderately (correlation coefficients typically 0.4-0.6) due to varied post-transcriptional and post-translational regulation. This biological variation is not experimental error but reflects complex regulatory mechanisms .

How should researchers interpret and validate bands of unexpected molecular weight when using At5g10810 Antibody?

Unexpected bands require systematic investigation before dismissal as non-specific binding:

• Potential biological explanations:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Alternative splicing variants

  • Proteolytic processing

  • Protein complexes resistant to denaturation

• Validation approaches:

  • Compare patterns across different tissues/conditions

  • Analyze knockout/knockdown samples

  • Perform mass spectrometry on immunoprecipitated bands

  • Use epitope-tagged versions of the protein

• Technical considerations:

  • Test different sample preparation methods

  • Vary denaturation conditions (temperature, reducing agents)

  • Use gradient gels for better resolution

  • Apply 2D electrophoresis to separate by both pI and molecular weight

• Quantitative assessment:

  • Compare band intensities under different conditions

  • Correlate with functional assays

Research indicates that approximately 15-25% of unexpected bands represent biologically relevant protein forms rather than non-specific binding. Careful validation can transform apparent artifacts into valuable discoveries about protein regulation and processing .

How can researchers effectively use At5g10810 Antibody for chromatin immunoprecipitation (ChIP) experiments?

Optimizing ChIP with At5g10810 Antibody requires specialized approaches for plant chromatin:

• Crosslinking optimization:

  • Test formaldehyde concentrations (0.75-2%)

  • Compare single vs. dual crosslinking (formaldehyde + disuccinimidyl glutarate)

  • Optimize crosslinking time (10-30 minutes)

• Chromatin preparation:

  • Cell-type specific nuclear isolation

  • Sonication parameters: amplitude, cycle number, duration

  • Target fragment size: 200-500 bp for standard ChIP, 50-300 bp for ChIP-seq

• Immunoprecipitation conditions:

  • Pre-clearing with protein A/G beads and non-specific IgG

  • Antibody amount: 2-5 μg per ChIP reaction

  • Incubation time: 4-16 hours at 4°C

  • Washing stringency gradient

• Controls essential for interpretation:

  • Input chromatin (non-immunoprecipitated)

  • IgG control (non-specific antibody)

  • Positive control regions (known binding sites)

  • Negative control regions (unexpressed genes)

• Data analysis considerations:

  • Normalization to input and IgG controls

  • Peak calling algorithms

  • Integration with transcriptome data

For ChIP-seq applications, additional quality control metrics include IP efficiency (>5%), library complexity (>80% unique reads), and signal-to-noise ratio (>3). Research shows that optimized ChIP protocols can achieve 20-30 fold enrichment of true binding sites over background .

What are the methodological considerations for using At5g10810 Antibody in super-resolution microscopy?

Super-resolution microscopy with At5g10810 Antibody requires specific optimization:

• Sample preparation considerations:

  • Fixation: Paraformaldehyde (2-4%) with low glutaraldehyde (0.1-0.2%)

  • Permeabilization: Optimize detergent type and concentration

  • Antigen retrieval: Test citrate buffer, EDTA, or enzymatic methods

  • Reducing autofluorescence: Sodium borohydride or Sudan Black B treatment

• Immunolabeling strategies:

  • Primary antibody: Extended incubation (overnight at 4°C)

  • Secondary antibody selection: F(ab')2 fragments for reduced size

  • Fluorophore selection: Photostability and quantum yield

  • Fluorophore density: Critical for STORM/PALM techniques

• Imaging parameters:

  • Buffer compositions for optimal fluorophore blinking

  • Laser power and exposure time optimization

  • Drift correction strategies

  • Sampling density (Nyquist criterion)

• Validation and controls:

  • Multi-color co-localization

  • Correlative light and electron microscopy

  • Quantitative analysis of spatial distribution

• Data analysis considerations:

  • Localization precision calculation

  • Cluster analysis methods

  • 3D reconstruction approaches

Super-resolution techniques can achieve 10-20 nm resolution compared to the 200-300 nm diffraction limit of conventional microscopy. Studies show that optimized super-resolution protocols can reveal previously undetectable subcellular distributions and co-localization patterns of plant proteins .

How can researchers apply At5g10810 Antibody for studying protein dynamics during plant stress responses?

Investigating protein dynamics during stress responses requires specialized approaches:

• Time-course experimental design:

  • Short-term kinetics: 5, 15, 30, 60, 120 minutes

  • Long-term adaptation: 6, 12, 24, 48, 72 hours

  • Recovery phases: Removal of stress and monitoring

• Subcellular fractionation techniques:

  • Differential centrifugation

  • Density gradient separation

  • Affinity purification of organelles

  • Sequential protein extraction

• Post-translational modification analysis:

  • Phospho-specific antibody generation

  • Phos-tag gels for mobility shift detection

  • 2D gel electrophoresis

  • Mass spectrometry validation

• Protein complex dynamics:

  • Blue native PAGE

  • Size exclusion chromatography

  • Crosslinking followed by immunoprecipitation

  • FRET-based interaction studies

• Single-cell approaches:

  • Laser capture microdissection

  • Flow cytometry of protoplasts

  • Live cell imaging with split fluorescent proteins

This methodological framework enables researchers to distinguish between rapid signaling events (occurring within minutes) and adaptive responses (occurring over hours to days). Research indicates that stress responses typically involve multiple waves of protein modification, relocalization, and abundance changes that cannot be captured at single timepoints .

What systematic validation approaches should researchers employ before using At5g10810 Antibody in critical experiments?

Comprehensive validation is essential before investing in extensive experiments:

• Genetic validation:

  • Knockout/knockdown lines: Signal should be reduced/absent

  • Overexpression lines: Signal should increase proportionally

  • Complementation lines: Signal should be restored

• Biochemical validation:

  • Western blot: Single band of expected molecular weight

  • Peptide competition: Signal should be blocked by immunizing peptide

  • Recombinant protein: Antibody should recognize purified protein

  • Mass spectrometry: Confirm identity of immunoprecipitated proteins

• Comparative validation:

  • Multiple antibodies against different epitopes

  • Correlation with fluorescent protein tagging

  • Agreement with known expression patterns

• Application-specific validation:

  • For IF/IHC: Absence of signal in knockout tissues

  • For IP: Enrichment of target in IP vs. input

  • For ChIP: Enrichment at known binding sites

• Quantitative assessment:

  • Titration curves for linearity of response

  • Standard curves with recombinant protein

  • Signal-to-noise ratio calculation

How can researchers distinguish between specific and non-specific binding in complex plant tissue samples?

Distinguishing specific from non-specific signals requires multiparametric approaches:

• Biological controls:

  • Signal absence/reduction in knockout/knockdown lines

  • Signal increase in overexpression systems

  • Tissue/cell type specificity matching known biology

• Technical controls:

  • Secondary antibody only

  • Isotype control antibody

  • Pre-immune serum

• Blocking strategies:

  • Pre-adsorption with immunizing peptide/protein

  • Increasing blocking reagent concentration

  • Testing different blocking agents

• Signal characteristics:

  • Reproducibility across experimental replicates

  • Consistency across different detection methods

  • Appropriate subcellular localization

• Response to experimental manipulation:

  • Changes in expected direction after treatment

  • Dose-dependent responses

  • Kinetics matching known biology

Researchers should create a validation matrix combining multiple approaches, as no single test can definitively establish specificity. Studies show that using three independent validation methods can reduce false positives by >90% compared to relying on a single validation approach .

What are the latest approaches for antibody validation in plant systems that researchers should consider?

Recent advances in antibody validation provide powerful new approaches:

• CRISPR/Cas9-based validation:

  • Generate precise gene knockouts

  • Create epitope modifications

  • Implement inducible degradation systems

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins

  • Compare antibody staining with biotin labeling patterns

  • Validate protein interaction networks

• Single-cell validation techniques:

  • Flow cytometry of protoplasts

  • Single-cell immunostaining with image cytometry

  • Correlation with single-cell transcriptomics

• Orthogonal validation methods:

  • RNA-protein correlation in different tissues/conditions

  • Targeted proteomics (PRM/MRM)

  • Nanobody-based detection systems

• Community resources:

  • Antibody validation repositories

  • Standard operating procedures

  • Open-source validation datasets

These advanced approaches address the limitations of traditional validation methods. For instance, CRISPR-based validation can confirm antibody specificity with precision that exceeds traditional genetic approaches, reducing false positive rates from approximately 30-40% to under 5%. Integrating multiple orthogonal validation approaches represents the current gold standard in the field .