At3g28410 Antibody

What is the At3g28410 protein and why is it significant in plant research?

At3g28410 encodes a putative F-box/LRR-repeat protein originally identified in Arabidopsis thaliana. This protein belongs to the diverse superfamily of F-box proteins, which function as substrate recognition components of SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complexes. These complexes play critical roles in protein ubiquitination and subsequent degradation via the 26S proteasome pathway.

The significance of At3g28410 extends beyond Arabidopsis, as homologous genes have been identified in other plant species including Solanum lycopersicum (tomato) and Nicotiana tomentosiformis, suggesting evolutionary conservation and potential functional importance across plant taxa . The protein appears to contain two F-box domains, indicating a potentially complex role in substrate recognition or regulatory mechanisms .

What techniques can be used to validate the specificity of an At3g28410 antibody?

To validate antibody specificity for At3g28410, researchers should implement a multi-pronged approach:

• Western blot analysis using positive and negative controls:

  • Positive control: Recombinant At3g28410 protein or extract from wildtype plants

  • Negative control: Protein extract from At3g28410 knockout lines like the SAIL_905_D05 T-DNA insertion line

• Immunoprecipitation followed by mass spectrometry:

  • Pull-down experiments with the antibody should identify peptides matching the expected sequence of At3g28410

  • Similar methodologies to those used in serum proteomics can be applied, including bottom-up mass spectrometry techniques

• Immunohistochemistry with genetic controls:

  • Compare signal patterns between wildtype and At3g28410 mutant tissues

  • Preabsorption of the antibody with purified antigen should eliminate specific signals

• Blocking experiments with recombinant protein:

  • Pre-incubation of the antibody with purified At3g28410 protein should prevent immunoreactivity

• Cross-reactivity testing:

  • Test against closely related F-box proteins to ensure specificity, particularly those identified in the same chromosomal region of Arabidopsis

What expression patterns have been documented for At3g28410 in different plant tissues?

Expression data for At3g28410 is limited, but examination of publicly available resources indicates low to moderate expression across multiple tissues. RT-PCR analysis can be implemented using primers targeting At3g28410 (forward: CCTTGTTGTGACAAGTCC, reverse: TGACATGTTCCCGAGGC) to detect tissue-specific expression patterns .

Protein expression should be confirmed using validated antibodies in conjunction with appropriate controls, particularly the SAIL_905_D05 T-DNA insertion line that has been confirmed to contain an insertion in exon 3 of the 3-exon gene . This knockout line serves as an excellent negative control for antibody specificity validation.

How can researchers distinguish between the potential multiple isoforms of At3g28410 using antibodies?

Distinguishing between potential At3g28410 isoforms requires carefully designed antibody epitope targeting:

• Isoform-specific epitope identification:

  • Analyze the mRNA and protein sequences of potential isoforms (e.g., alternative splice variants)

  • Design antibodies against unique regions not shared between isoforms

• 2D gel electrophoresis followed by western blotting:

  • Separate proteins by both molecular weight and isoelectric point

  • Identify distinct spots representing different isoforms using the antibody

• Immunoprecipitation coupled with isoform-specific PCR:

  • Pull down the protein using the antibody

  • Analyze associated mRNAs to determine which isoforms are present

• Mass spectrometry analysis post-immunoprecipitation:

  • Detect isoform-specific peptides using techniques similar to those employed in serum proteomics studies

  • Implement bottom-up mass spectrometry to identify unique peptide signatures

• Comparative analysis between wildtype and transgenic plants:

  • Generate plants expressing single isoforms of At3g28410

  • Use antibodies to determine migration patterns and expression levels of each isoform

What are the challenges in developing antibodies against plant F-box proteins like At3g28410?

Developing antibodies against plant F-box proteins presents several unique challenges:

• Structural complexity and conservation:

  • F-box domains show high conservation across family members, increasing cross-reactivity risk

  • At3g28410 contains both F-box and LRR repeats, creating potential epitope masking

  • The protein appears to contain two F-box domains, further complicating unique epitope selection

• Post-translational modifications:

  • F-box proteins often undergo regulatory phosphorylation and ubiquitination

  • Modifications can mask epitopes or alter antibody recognition

• Protein-protein interactions:

  • F-box proteins function in SCF complexes, potentially obscuring antibody access sites

  • When bound to substrates, key epitopes may be unavailable

• Low endogenous expression:

  • Expression of At3g28410 may be tissue-specific or condition-dependent

  • No clear expression data is available in the provided resources, suggesting potentially low abundance

• Protein stability and degradation:

  • As components of degradation machinery, F-box proteins may undergo rapid turnover

  • Sample preparation must minimize degradation while maintaining native conformation

How can interaction partners of At3g28410 be identified using antibody-based approaches?

To identify At3g28410 interaction partners, researchers can employ several antibody-dependent techniques:

• Co-immunoprecipitation (Co-IP) followed by mass spectrometry:

  • Use anti-At3g28410 antibodies to pull down the protein complex

  • Identify associated proteins via LC-MS/MS similar to techniques used in serum proteomics

  • Compare results against negative controls (ideally the SAIL_905_D05 T-DNA insertion line)

• Proximity-based labeling coupled with immunoprecipitation:

  • Fuse At3g28410 with a proximity labeling enzyme (BioID or APEX)

  • Use anti-At3g28410 antibodies to confirm expression and proper localization

  • Identify labeled proteins in the vicinity of At3g28410

• Yeast two-hybrid validation:

  • Screen for potential interactors using Y2H

  • Validate interactions in planta using Co-IP with anti-At3g28410 antibodies

  • Confirm biological relevance through functional assays

• Antibody-based protein arrays:

  • Probe plant protein arrays with purified At3g28410

  • Validate candidate interactors using reciprocal Co-IP with anti-At3g28410 antibodies

A comprehensive interaction map should include analysis of both structural and functional SCF complex components, including Skp1, Cullin, and Rbx proteins, as well as potential substrates targeted by the At3g28410 F-box protein.

What is the optimal immunogen design strategy for generating antibodies against At3g28410?

Optimal immunogen design for At3g28410 antibody production requires careful sequence analysis and structural consideration:

• Epitope selection criteria:

  • Target unique regions not conserved in other F-box proteins

  • Avoid the conserved F-box domain (approximately 50 amino acids) to prevent cross-reactivity

  • Focus on the more variable LRR-repeat regions unique to At3g28410

  • Consider surface accessibility based on structural predictions

• Immunogen formats:

  • Recombinant protein fragments: Express protein domains excluding the F-box domain

  • Synthetic peptides: 15-20 amino acids from unique regions with terminal cysteine for conjugation

  • Full-length protein: Express full At3g28410 protein (as available from Genscript, catalog OSo82292)

• Expression system considerations:

  • Bacterial systems (E. coli): Suitable for producing denatured protein fragments

  • Insect cell systems: Better for obtaining properly folded full-length protein

  • Plant expression systems: Optimal for preserving plant-specific post-translational modifications

• Conjugation and presentation:

  • Carrier protein conjugation (KLH or BSA) for small peptides

  • Fusion with solubility tags (MBP, GST) for recombinant fragments

  • Purification tags that can be cleaved post-purification

• Validation of immunogen:

  • Confirm sequence integrity by mass spectrometry

  • Verify folding status using circular dichroism if conformation-specific antibodies are desired

How should researchers optimize immunohistochemistry protocols for detecting At3g28410 in plant tissues?

Optimizing immunohistochemistry for At3g28410 detection requires:

• Tissue preparation considerations:

  • Fixation method: Compare paraformaldehyde (for protein crosslinking) vs. alcohol-based (for morphology)

  • Embedding medium: Paraffin for thin sections vs. cryo-embedding for antigen preservation

  • Antigen retrieval: Test heat-induced (citrate buffer, pH 6.0) and enzymatic methods

• Blocking and antibody incubation:

  • Blocking agents: Compare BSA, normal serum, and commercial blockers

  • Primary antibody dilution series: Test 1:100 to 1:1000 to optimize signal-to-noise ratio

  • Incubation conditions: 4°C overnight vs. room temperature for shorter periods

  • Secondary antibody selection: Species-specific, highly cross-adsorbed antibodies

• Signal development and detection:

  • Chromogenic detection: DAB or NBT/BCIP with appropriate enzyme-conjugated secondaries

  • Fluorescent detection: Alexa Fluor or similar stable fluorophores

  • Signal amplification: Consider tyramide signal amplification for low-abundance targets

• Controls and validation:

  • Positive control: Tissues with known At3g28410 expression

  • Negative control: SAIL_905_D05 T-DNA insertion line tissues

  • Primary antibody omission control

  • Competing peptide control: Pre-incubation with immunizing peptide

• Colocalization studies:

  • Double-labeling with markers for cellular compartments

  • Co-staining with antibodies against known interaction partners

What considerations are important when using At3g28410 antibodies for protein quantification?

When using antibodies for At3g28410 quantification, researchers should address:

• Sample preparation standardization:

  • Tissue collection: Standardize developmental stage and environmental conditions

  • Protein extraction: Optimize buffer composition for F-box protein solubilization

  • Protein determination: Use methods resistant to buffer interference (BCA, Bradford)

  • Sample handling: Minimize freeze-thaw cycles to prevent degradation

• Quantification method selection:

  • Western blotting with densitometry: Semi-quantitative with appropriate loading controls

  • ELISA: Develop sandwich ELISA using two antibodies targeting different epitopes

  • Capillary electrophoresis with immunodetection: Higher sensitivity for low abundance proteins

• Standard curve generation:

  • Recombinant protein standards: Use purified At3g28410 protein

  • Reference sample: Include a common reference sample across all experiments

  • Linear range determination: Establish the quantitative range of the assay

• Normalization strategies:

  • Housekeeping proteins: Use stable reference proteins (actin, tubulin)

  • Total protein normalization: Methods like Ponceau S or SYPRO Ruby staining

  • Spike-in controls: Add known amounts of tagged recombinant protein

• Statistical considerations:

  • Technical replicates: Minimum of three per biological sample

  • Biological replicates: Minimum of three independent experiments

  • Appropriate statistical tests for the specific experimental design

How can researchers interpret contradictory results between antibody-based methods and genomic/transcriptomic data for At3g28410?

When facing contradictions between antibody-based protein detection and genomic/transcriptomic data:

• Methodological reconciliation approach:

  • Verify antibody specificity using knockout lines (e.g., SAIL_905_D05)

  • Confirm transcript presence/absence using RT-PCR with validated primers

  • Sequence verification to rule out genetic variation between experimental lines

• Post-transcriptional regulation assessment:

  • Investigate microRNA-mediated regulation of At3g28410

  • Examine RNA stability using actinomycin D chase experiments

  • Assess alternative splicing using RT-PCR with primers spanning potential splice junctions

• Post-translational regulation analysis:

  • Measure protein stability using cycloheximide chase experiments

  • Investigate ubiquitination status of At3g28410 itself

  • Assess protein localization versus transcript distribution

• Technical considerations:

  • Re-evaluate primer specificity and antibody validation

  • Consider detection threshold differences between methods

  • Investigate potential environment-dependent expression patterns

• Integrated data analysis approach:

  • Correlate protein levels with transcript abundance across conditions

  • Develop mathematical models accounting for transcription, translation, and degradation rates

  • Consider using mass spectrometry as an antibody-independent protein detection method

What statistical approaches are most appropriate for analyzing At3g28410 protein expression across different experimental conditions?

Appropriate statistical analyses for At3g28410 protein expression data include:

• Descriptive statistics:

  • Central tendency: Mean, median for expression levels

  • Dispersion: Standard deviation, standard error, coefficient of variation

  • Data visualization: Box plots, violin plots for distribution patterns

• Inferential statistics for group comparisons:

  • Parametric tests: t-test (two groups), ANOVA (multiple groups) if normality assumptions are met

  • Non-parametric alternatives: Mann-Whitney U (two groups), Kruskal-Wallis (multiple groups)

  • Post-hoc tests: Tukey's HSD, Bonferroni, or Dunnett's tests for multiple comparisons

• Correlation and regression analyses:

  • Pearson/Spearman correlation: Relationship between At3g28410 levels and physiological parameters

  • Linear regression: Predicting protein levels based on experimental variables

  • Multiple regression: Accounting for multiple experimental factors

• Time-series analysis methods:

  • Repeated measures ANOVA: For time-dependent expression changes

  • Mixed-effects models: Accounting for both fixed and random effects

  • Time-course trend analysis: Identifying patterns in expression dynamics

• Multivariate analysis approaches:

  • Principal Component Analysis: Reducing dimensionality of complex datasets

  • Cluster analysis: Identifying groups of conditions with similar expression patterns

  • Path analysis: Evaluating causal relationships between variables

How should researchers address potential artifacts in Co-IP experiments using At3g28410 antibodies?

To address potential artifacts in Co-IP experiments with At3g28410 antibodies:

• Pre-experiment validation:

  • Confirm antibody specificity using western blotting against wildtype and knockout tissues

  • Determine optimal antibody concentration to minimize non-specific binding

  • Test different lysis and IP buffer compositions to maintain complex integrity

• Appropriate controls:

  • Negative control: IP with non-specific IgG from the same species

  • Knockout control: IP from SAIL_905_D05 T-DNA line samples

  • Input control: Analysis of pre-IP sample to confirm target presence

  • Reciprocal IP: Confirm interactions by pulling down with antibodies against suspected partners

• Stringency optimization:

  • Buffer salt concentration: Test series from 150-500 mM NaCl

  • Detergent type and concentration: Compare NP-40, Triton X-100, and CHAPS

  • Wash number and duration: Balance between removing non-specific binders and maintaining complexes

• Crosslinking considerations:

  • Reversible crosslinkers: DSP, DTBP for stabilizing transient interactions

  • Formaldehyde crosslinking: For capturing in vivo complexes

  • Crosslinking titration: Determine optimal concentration to prevent artifacts

• Validation techniques:

  • Mass spectrometry identification of co-immunoprecipitated proteins

  • Western blot confirmation of specific interaction partners

  • Functional validation through genetic or pharmacological perturbation

What quality control measures should be implemented when using commercial At3g28410 antibodies?

Quality control for commercial At3g28410 antibodies should include:

• Initial validation:

  • Western blot against recombinant At3g28410 protein (available from GenScript, catalog OSo82292)

  • Comparison with knockout line (SAIL_905_D05) tissue extracts

  • Peptide competition assay using the immunizing antigen

  • Cross-reactivity testing against related F-box proteins

• Lot-to-lot consistency testing:

  • Side-by-side testing of old and new antibody lots

  • Standardized positive control samples for comparison

  • Record key parameters: detection sensitivity, background, banding pattern

• Storage and handling validation:

  • Freeze-thaw stability assessment

  • Temperature sensitivity testing

  • Carrier protein addition evaluation for dilute antibody solutions

• Application-specific validation:

  • For western blotting: Optimize blocking agents and dilution ratios

  • For immunoprecipitation: Test bead types and binding conditions

  • For immunohistochemistry: Compare fixation methods and antigen retrieval techniques

• Documentation and record-keeping:

  • Maintain detailed records of validation experiments

  • Document optimal working conditions for each application

  • Create standard operating procedures for each antibody

What are the most common causes of false positive and false negative results when using At3g28410 antibodies, and how can they be mitigated?

Common causes of false results and mitigation strategies include:

False Positives:

• Cross-reactivity with related proteins:

  • Mitigation: Use peptide competition assays to confirm specificity

  • Mitigation: Include knockout controls (SAIL_905_D05)

  • Mitigation: Perform immunoprecipitation with subsequent mass spectrometry to identify all bound proteins

• Non-specific binding:

  • Mitigation: Optimize blocking (5% milk, 3% BSA, or commercial blockers)

  • Mitigation: Increase washing stringency (higher salt, mild detergents)

  • Mitigation: Pre-clear lysates with beads alone before immunoprecipitation

• Secondary antibody issues:

  • Mitigation: Include secondary-only controls

  • Mitigation: Use highly cross-adsorbed secondary antibodies

  • Mitigation: Consider direct conjugation of primary antibody

• Endogenous peroxidase/phosphatase activity:

  • Mitigation: Include blocking steps specific to these enzymes

  • Mitigation: Use fluorescent detection methods instead of enzymatic

  • Mitigation: Incorporate appropriate inhibitors in sample preparation

False Negatives:

• Epitope masking or modification:

  • Mitigation: Test multiple antibodies targeting different epitopes

  • Mitigation: Try different antigen retrieval methods

  • Mitigation: Use denaturing conditions for western blotting

• Protein degradation:

  • Mitigation: Include protease inhibitor cocktails

  • Mitigation: Minimize sample processing time

  • Mitigation: Keep samples cold throughout preparation

• Low abundance protein:

  • Mitigation: Enrich samples through immunoprecipitation or fractionation

  • Mitigation: Use signal amplification methods

  • Mitigation: Increase sample loading within linear range

• Technical factors:

  • Mitigation: Optimize protein transfer conditions

  • Mitigation: Verify antibody functionality with positive controls

  • Mitigation: Consider protein extraction methods optimized for membrane proteins

How can researchers troubleshoot inconsistent results when comparing At3g28410 antibody data across different plant species?

When troubleshooting cross-species antibody reactivity issues:

• Sequence homology analysis:

  • Perform protein sequence alignment between At3g28410 and putative homologs in target species

  • Focus on epitope regions to predict cross-reactivity

  • Compare F-box/LRR-repeat protein At3g28410 sequences from Arabidopsis, tomato, and tobacco

• Epitope conservation verification:

  • Design synthetic peptides matching the homologous regions from each species

  • Test antibody reactivity against these peptides via ELISA

  • Perform competition assays using species-specific peptides

• Extraction protocol optimization:

  • Compare protein extraction methods optimized for each species

  • Test different detergents and buffer compositions

  • Adjust extraction protocols based on tissue-specific characteristics

• Experimental validation approaches:

  • Use recombinant proteins from each species as positive controls

  • Include genetic controls (overexpression and knockout) where available

  • Perform mass spectrometry to confirm protein identity in immunoprecipitates

• Cross-species normalization strategies:

  • Develop species-specific standard curves using recombinant proteins

  • Include conserved reference proteins as internal controls

  • Consider using conserved epitope tags in transgenic approaches to ensure consistent detection