At1g47340 Antibody

What is the At1g47340 gene and what protein does it encode?

At1g47340 encodes an F-box protein called PHIF1 (PHR1 Interacting F-box protein 1) in Arabidopsis thaliana. It functions as a component of SCF complexes that determine the stability of the PHR1 transcription factor, which is critical for phosphate starvation responses. The protein contains characteristic F-box domains that facilitate interaction with SKP-1 proteins to form functional E3 ubiquitin ligase complexes for targeted protein degradation .

When designing experiments to study this protein, researchers should consider its relatively low expression levels and tissue-specific regulation patterns. Quantitative analyses typically require sensitive detection methods since baseline expression may be modest until induced by specific environmental conditions such as arsenate exposure.

How do At1g47340 antibodies differ from other plant protein antibodies in terms of specificity?

At1g47340 antibodies require particular attention to specificity due to the large F-box protein family in Arabidopsis (hundreds of F-box genes are encoded in the genome) . Quality antibodies must be raised against unique epitopes that do not cross-react with other F-box family members.

When validating antibody specificity, researchers should:

• Perform western blots using recombinant At1g47340 protein as a positive control

• Include knockout mutant samples as negative controls

• Test for cross-reactivity with closely related F-box proteins

• Validate with orthogonal methods such as mass spectrometry identification

Antibody specificity can be confirmed by correlating protein detection with transcript levels from qRT-PCR analyses under conditions known to affect At1g47340 expression, such as arsenate treatments .

What are the recommended storage and handling conditions for At1g47340 antibodies?

For optimal performance of At1g47340 antibodies, follow these evidence-based handling protocols:

• Storage temperature: Maintain antibody aliquots at -80°C for long-term storage and at -20°C for working stocks

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

• Use glycerol (50%) for storage buffers to prevent freeze-thaw damage

• Working dilutions should be prepared fresh and stored at 4°C for no more than 2 weeks

• Always centrifuge antibody solutions briefly before use to pellet any aggregates

Quality control testing should include periodic validation against positive controls to ensure continued specificity and sensitivity, particularly when using antibodies for quantitative analyses of At1g47340 expression in response to arsenate or other stress conditions .

How can At1g47340 antibodies be optimized for Western blot analysis of plant samples?

Optimizing At1g47340 antibody use for Western blot requires special consideration due to the protein's regulatory nature and potential rapid turnover. Based on research evidence:

• Extraction buffer composition:

  • Include proteasome inhibitors (e.g., MG132) to prevent degradation

  • Add phosphatase inhibitors to preserve post-translational modifications

  • Use fresh reducing agents to maintain epitope accessibility

• Recommended protocol adjustments:

  • Longer transfer times (overnight at lower voltage) improve detection of membrane-associated fractions

  • BSA blocking (3-5%) typically provides better results than milk-based blockers

  • Extended primary antibody incubation (overnight at 4°C) enhances sensitivity

Research has shown that the detection of At1g47340 protein can be challenging as its levels fluctuate in response to arsenate treatment, with protein degradation occurring within 3-6 hours of exposure and subsequent re-stabilization after 24 hours . These temporal dynamics should inform sampling timepoints when designing experiments.

What controls are essential when using At1g47340 antibodies for co-immunoprecipitation studies?

For co-immunoprecipitation (Co-IP) studies investigating At1g47340 interactions, particularly with SKP-1 proteins (AT5G42190), the following controls are critical:

• Input controls: Reserve 5-10% of pre-immunoprecipitation lysate to confirm target protein presence

• Negative controls:

  • IgG isotype control to detect non-specific binding

  • Immunoprecipitation from knockout/knockdown lines of At1g47340

  • Reciprocal IP using antibodies against expected interacting partners

• Competition controls: Pre-incubation with the immunizing peptide to demonstrate specificity

When investigating At1g47340 interactions with SKP-1, researchers should consider that these interactions may be transient and condition-specific. Evidence suggests that environmental conditions significantly affect the interaction specificity between F-box proteins and their partners . Therefore, experimental conditions should closely mimic the physiological context of interest.

How can immunohistochemistry with At1g47340 antibodies be optimized for plant tissue sections?

For immunohistochemistry applications with At1g47340 antibodies in plant tissues, consider these research-validated approaches:

• Fixation protocol optimization:

  • 4% paraformaldehyde typically preserves At1g47340 epitopes while maintaining tissue structure

  • Limited fixation time (2-4 hours) prevents epitope masking

  • Gentle permeabilization with 0.1-0.3% Triton X-100 improves antibody penetration

• Antigen retrieval methods:

  • Citrate buffer (pH 6.0) heat-induced epitope retrieval works well for many plant F-box proteins

  • Enzymatic retrieval with proteinase K can be effective but requires careful titration

• Signal amplification:

  • Tyramide signal amplification can enhance detection of low-abundance At1g47340

  • Quantum dots as secondary antibody labels provide superior photostability for confocal imaging

Studies examining At1g47340's role in arsenate response mechanisms have shown that protein localization patterns may change upon stress treatment, with potential translocation between cellular compartments . Therefore, careful attention to physiological conditions prior to fixation is essential.

How should researchers interpret contradictory results between transcript and protein levels of At1g47340?

Discrepancies between At1g47340 transcript and protein levels are common and physiologically relevant. Research shows that:

• Transcript-protein discordance mechanisms:

  • Post-transcriptional regulation via microRNAs or RNA-binding proteins

  • Post-translational modifications affecting protein stability

  • Targeted protein degradation via the ubiquitin-proteasome system

• Analysis approach:

  • Examine temporal dynamics: Transcript levels may precede protein accumulation

  • Consider half-life differences: At1g47340 protein turnover may be faster than transcript degradation

  • Integrate multiple timepoints: Single timepoint analyses often miss dynamic regulation

Published data demonstrate that while PHR1 transcript accumulation was not altered after 3 hours of arsenate treatment, there was significant protein degradation, indicating post-translational regulation . After 24 hours, both transcript and protein levels increased, suggesting coordinated transcriptional and post-translational regulation during recovery phases.

What statistical approaches are recommended for quantifying At1g47340 protein expression changes across experimental conditions?

Based on published methodologies for analyzing At1g47340 and similar regulatory proteins:

• Recommended quantification methods:

  • Normalize to multiple loading controls (actin and total protein stains)

  • Use relative fold change rather than absolute intensity values

  • Apply ANOVA with post-hoc tests for multi-condition comparisons

• Statistical considerations:

  • Account for non-normal distributions using appropriate transformations

  • Include biological replicates (n≥3) rather than technical replicates alone

  • Apply false discovery rate corrections for multiple comparisons

• Data presentation standards:

  • Include representative blot images alongside quantification

  • Report adjusted relative intensity values normalized to controls

  • Provide clear description of normalization methods

In published studies, researchers normalized At1g47340-related protein signals to actin controls and expressed values relative to untreated conditions. Statistical significance was typically established using Student's t-test with p<0.05 considered significant .

How can researchers distinguish between specific At1g47340 signal and background in immunofluorescence studies?

Differentiating specific At1g47340 signal from background in immunofluorescence requires systematic controls and analysis:

• Essential controls:

  • Secondary-only controls to establish baseline autofluorescence

  • Pre-immune serum controls to identify non-specific binding

  • Peptide competition assays to confirm epitope specificity

  • Negative control tissues (knockout/knockdown lines)

• Analytical approaches:

  • Signal-to-noise ratio calculations across multiple fields of view

  • Colocalization with known compartment markers to confirm expected localization

  • Comparison of signal intensity distribution between experimental and control samples

• Advanced techniques:

  • Spectral unmixing to separate At1g47340 signal from autofluorescence

  • Fluorescence lifetime imaging to distinguish specific binding

  • Super-resolution microscopy to improve signal discrimination

When studying At1g47340 in plant tissues, researchers should be particularly attentive to changes in subcellular localization upon arsenate or arsenite treatment, as confocal analyses have shown internalization of related proteins into vesicles following treatment .

How can At1g47340 antibodies be used to study arsenate response mechanisms in plants?

At1g47340 antibodies provide powerful tools for investigating arsenate response mechanisms:

• Experimental design considerations:

  • Temporal sampling: Collect samples at 0, 3, 6, and 24 hours post-treatment to capture dynamic responses

  • Concentration gradients: Test multiple arsenate concentrations (30μM, 100μM) to determine dose-dependent effects

  • Tissue specificity: Separate analysis of roots versus shoots to identify tissue-specific responses

• Analytical approaches:

  • Protein stability assays: Monitor At1g47340 accumulation in response to arsenate

  • Interaction studies: Examine associations with PHR1 and SKP-1 components

  • Subcellular fractionation: Track potential relocalization during stress response

Research has demonstrated that arsenate exposure triggers a coordinated response involving PHR1 degradation and subsequent recovery after 24 hours, with At1g47340 playing a critical role in this regulatory process . This temporal dynamic is essential for adapting phosphate/arsenate uptake to the plant's arsenic detoxification capacity.

What experimental design is recommended for studying At1g47340 protein interactions with SKP-1 under different environmental conditions?

To effectively study condition-specific interactions between At1g47340 and SKP-1:

• Recommended experimental design:

  • Split-plot design with environmental conditions as main plots

  • Technical approaches as sub-plots to control for variability

  • Minimum three biological replicates per condition

• Environmental variables to consider:

  • Arsenate/arsenite concentrations (30-100μM range)

  • Phosphate availability (deficient vs. sufficient)

  • Temporal dynamics (early response vs. acclimation phase)

• Interaction analysis methods:

  • Co-immunoprecipitation with both forward and reverse approaches

  • Proximity ligation assays for in situ interaction detection

  • FRET/FLIM analysis for real-time interaction monitoring

Research has shown that interactions between F-box proteins and SKP-1 are highly specific and environmentally regulated. For At1g47340 specifically, understanding under which environmental conditions these interactions occur is critical for elucidating their biological significance .

How can At1g47340 antibodies be used in chromatin immunoprecipitation experiments to study transcriptional regulation?

While At1g47340 itself is not a transcription factor, its role in regulating PHR1 stability makes it relevant for chromatin studies:

• Experimental approach:

  • Tandem chromatin immunoprecipitation (re-ChIP) to capture At1g47340-PHR1-DNA complexes

  • ChIP-seq to identify genome-wide PHR1 binding sites affected by At1g47340

  • CUT&RUN for higher resolution and lower background in At1g47340-associated chromatin

• Technical considerations:

  • Crosslinking optimization: Brief formaldehyde fixation (1-2 minutes) to capture transient interactions

  • Sonication parameters: Shorter fragments (200-300bp) improve resolution

  • Antibody validation: Confirm specificity in ChIP-grade applications

• Data analysis:

  • Integration with transcriptomics to correlate binding with expression changes

  • Motif enrichment analysis to identify DNA elements in At1g47340-regulated genes

  • Comparison across environmental conditions to detect stress-specific regulation

Studies have demonstrated that PHR1 binding to target promoters is dynamically regulated during arsenate stress, with At1g47340 playing a crucial role in this process through modulation of PHR1 stability .

What are the most common issues when working with At1g47340 antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with At1g47340 antibodies:

• Low signal intensity:

  • Cause: Low endogenous expression or epitope masking

  • Solution: Use signal amplification methods or optimize extraction conditions with proteasome inhibitors

• Multiple bands on Western blots:

  • Cause: Post-translational modifications or partial degradation

  • Solution: Include phosphatase inhibitors and fresh protease inhibitors in extraction buffers

• Variable results between experiments:

  • Cause: Environmental condition variation affecting At1g47340 expression

  • Solution: Strictly control growth conditions and harvest timing, especially in relation to light cycles

• Background in immunofluorescence:

  • Cause: Cross-reactivity with related F-box proteins

  • Solution: Pre-absorb antibody with plant extract from At1g47340 knockout lines

Evidence shows that At1g47340 protein levels change dramatically in response to arsenate treatment, with degradation after 3-6 hours followed by re-accumulation after 24 hours . This temporal variability must be considered when troubleshooting inconsistent results.

What validation methods confirm the specificity of At1g47340 antibodies in different experimental contexts?

Comprehensive validation of At1g47340 antibodies should include:

• Genetic validation approaches:

  • Testing in knockout/knockdown lines (negative control)

  • Complementation lines with tagged At1g47340 (positive control)

  • Overexpression lines to confirm signal intensity correlation

• Biochemical validation methods:

  • Western blot against recombinant At1g47340 protein

  • Peptide competition assays using immunizing peptide

  • Mass spectrometry identification of immunoprecipitated proteins

• Orthogonal confirmation techniques:

  • Correlation between protein detection and mRNA levels

  • Agreement between antibody-based methods and fluorescent protein fusions

  • Consistency with known biological responses (e.g., arsenate treatment)

Research demonstrates that validation using multiple approaches is essential due to the large family of F-box proteins in Arabidopsis and their potential for environmental condition-specific expression patterns .

How can researchers accurately quantify At1g47340 protein levels in plant tissues with high background or interfering compounds?

For accurate quantification in challenging plant samples:

• Sample preparation optimization:

  • Use polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds

  • Implement TCA/acetone precipitation to concentrate proteins and remove interfering compounds

  • Consider phase partitioning methods to enrich membrane-associated fractions

• Advanced quantification methods:

  • Selected reaction monitoring (SRM) mass spectrometry for absolute quantification

  • Capillary Western immunoassay (Wes/Jess systems) for higher sensitivity

  • ELISA development with sandwich approach for improved specificity

• Normalization strategies:

  • Multiple reference proteins (actin, tubulin, and GAPDH)

  • Total protein normalization with stain-free technology

  • Spike-in controls with known quantities of recombinant protein

Published studies examining At1g47340-related proteins demonstrated successful quantification using immunoblot analyses with actin hybridization as a loading control, reporting adjusted relative intensity of bands expressed relative to control conditions and normalized to corresponding loading controls .