At3g44130 Antibody

What is the At3g44130 protein and what are its key functional characteristics?

At3g44130 (also known as CEG or CEGENDUO) encodes an F-box and associated interaction domains-containing protein in Arabidopsis thaliana. It belongs to the S-LOCUS F-BOX (SFL) family and is also referenced as ATSFL61 or SFL61 . F-box proteins typically function as components of SCF ubiquitin-ligase complexes, where they play roles in substrate recognition for targeted protein degradation. The protein is involved in multiple cellular processes including protein-protein interactions and cellular signaling pathways.

What sample preparation methods are recommended for At3g44130 antibody experiments?

For optimal results with At3g44130 antibody experiments, the following sample preparation protocol is recommended:

• Harvest tissue from 7-day-old Arabidopsis seedlings (wild-type and appropriate mutant controls)

• Extract total protein using a buffer containing:

  • 50 mM HEPES-KOH buffer

  • 250 mM sucrose

  • 5% glycerol

  • 50 mM NaPP (sodium pyrophosphate)

  • 1 mM NaMo (sodium molybdate)

  • 25 mM NaF (sodium fluoride)

  • 10 mM EDTA

  • 0.5% PVP (polyvinylpyrrolidone)

  • 3 mM DTT (dithiothreitol)

  • 1 mM PMSF (phenylmethylsulfonyl fluoride)

  • 10 μM Leupeptin

  • 10 nM Calyculin

• For subcellular fractionation: centrifuge at 100,000 × g for 30 minutes at 4°C to separate soluble (S100) and microsomal (P100) fractions

• Denature protein samples at 65°C for 5 minutes before SDS-PAGE loading

What are the recommended controls for At3g44130 antibody experiments?

When conducting experiments with At3g44130 antibodies, the following controls should be included:

Positive controls:

• Wild-type Arabidopsis thaliana tissue (Col-0 ecotype)

• Recombinant At3g44130 protein (if available)

Negative controls:

• Null mutant tissue (knockout/knockdown lines for At3g44130)

• Pre-immune serum controls for polyclonal antibodies

• Secondary antibody-only controls

• Non-expressing tissue (if At3g44130 shows tissue-specific expression)

Similar to approaches used with other Arabidopsis proteins, include cytosolic marker proteins (for fractionation experiments) and housekeeping proteins (e.g., actin or GAPDH) as loading controls .

How should antibody dilutions be optimized for At3g44130 detection?

Optimal antibody dilutions should be determined empirically through titration experiments:

For Western blot optimization, follow protocols similar to those used for other Arabidopsis proteins, using 30 μg of total protein per lane and blocking with PBS-T + 5% milk for 1 hour at room temperature .

What strategies are most effective for validating At3g44130 antibody specificity?

For rigorous validation of At3g44130 antibodies, a multi-method approach is recommended:

Orthogonal validation:
Compare protein detection with RNA expression data. Analyze the correlation between antibody staining patterns and RNA-seq or microarray data for At3g44130 across different tissues and conditions. A high Kendall rank correlation indicates good antibody specificity .

Independent antibody validation:
Use multiple antibodies targeting non-overlapping regions of At3g44130. The antibodies should show similar staining patterns in terms of:

• Cell-type specificity

• Spatial distribution (cell-to-cell variability)

• Subcellular localization

Genetic validation:
Test the antibody on knockout/knockdown lines for At3g44130. A specific antibody should show significantly reduced or absent signal in these lines .

Recombinant protein controls:
Express and purify the At3g44130 protein or its domains in a heterologous system (e.g., E. coli) and confirm antibody recognition via Western blot .

For enhanced validation, perform epitope mapping to determine the exact binding region of the antibody, which helps predict potential cross-reactivity with related proteins .

What are the optimal methods for detecting At3g44130 interactions with other proteins?

To investigate protein interactions of At3g44130, several complementary approaches are recommended:

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

• Express At3g44130-GFP fusion protein in Arabidopsis (under a native or 35S promoter)

• Perform immunoprecipitation using GFP antibodies

• Process samples using either:

  • On-bead trypsin digestion

  • In-gel trypsin digestion

• Analyze using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

• Include appropriate controls (GFP-only expressing lines)

Validation criteria for interacting proteins:

• Enrichment compared to control samples

• Normalized spectral abundance factor (NSAF) values

• Multiple peptide identification

• Reproducibility across independent experiments

For proteins showing interaction with At3g44130, further validate using:

• Reciprocal co-IP experiments

• Bimolecular fluorescence complementation (BiFC)

• Yeast two-hybrid assays

• In vitro binding assays with purified components

How does subcellular localization affect At3g44130 antibody detection and what techniques are optimal for localization studies?

The subcellular localization of At3g44130 is critical for both experimental design and data interpretation. For comprehensive localization studies, employ multiple complementary techniques:

Western blot with subcellular fractionation:

• Perform differential centrifugation to separate:

  • Total protein (T)

  • Cytosolic fraction (C)

  • Microsomal fraction (P)

  • Nuclear fraction (N)

• Run SDS-PAGE followed by Western blot with At3g44130-specific antibodies

• Compare distribution patterns with known compartment markers

Fluorescent protein fusion approaches:

• Generate N- and C-terminal fusions (e.g., GFP::At3g44130 and At3g44130::GFP)

• Express in Arabidopsis under native or 35S promoter

• Validate expression using Northern blot and Western blot analysis

• Analyze by confocal microscopy in:

  • Intact tissues

  • Isolated protoplasts (to distinguish plasma membrane from cytosolic localization)

• Include appropriate controls (unfused GFP)

Immuno-electron microscopy:

• Fix Arabidopsis roots and leaves in 4% paraformaldehyde and 0.5% glutaraldehyde

• Embed in LR white resin

• Section (90 nm) and mount on formvar-coated slotted grids

• Block with TTBS containing 1% fish skin gelatin and 1% BSA

• Incubate with At3g44130 antibodies (1:50 dilution)

• Apply 10 nm gold-conjugated secondary antibodies

• Stain with uranyl acetate and lead citrate

• Visualize using electron microscopy

What challenges exist in detecting post-translational modifications of At3g44130 and how can they be addressed?

Detection of post-translational modifications (PTMs) on At3g44130 presents several challenges:

Challenges:

• Modification-specific antibodies for At3g44130 are rarely available

• PTMs may be substoichiometric or transient

• PTMs can affect antibody epitope recognition

• Sample preparation may cause PTM loss

Methodological solutions:

For comprehensive PTM analysis:

• Immunoprecipitate At3g44130 from tissues treated with PTM-inducing conditions

• Process for mass spectrometry analysis

• Search MS data for known PTM mass shifts

• Validate findings using site-directed mutagenesis of modified residues

How can immunoprecipitation protocols be optimized for At3g44130 in challenging Arabidopsis tissues?

Optimizing immunoprecipitation of At3g44130 from recalcitrant Arabidopsis tissues requires specific adaptations:

For tissues with high secondary metabolite content:

• Add 2% PVPP (polyvinylpolypyrrolidone) to extraction buffer

• Include 1% β-mercaptoethanol to prevent oxidation

• Add activated charcoal (0.1% w/v) during initial extraction

• Consider a pre-clearing step with non-specific IgG

For tissues with low At3g44130 expression:

• Scale up starting material (up to 5-10g tissue)

• Use gentler extraction conditions (lower detergent concentrations)

• Extend antibody incubation time (overnight at 4°C)

• Consider crosslinking approaches to stabilize transient interactions:

  • DSP (dithiobis[succinimidyl propionate], membrane-permeable)

  • Formaldehyde (1%, 10 min, quench with 125 mM glycine)

Optimized IP procedure:

• Extract proteins under conditions that maintain native interactions

• Pre-clear lysate with Protein A/G beads

• Incubate with At3g44130 antibody (2-5 μg) overnight at 4°C

• Add fresh Protein A/G beads for 3 hours

• Wash stringently (at least 4-5 washes with increasing salt concentration)

• Elute specifically (either with peptide competition or low pH)

• Analyze by Western blot or mass spectrometry

What are the optimal immunofluorescence techniques for At3g44130 localization in plant tissues?

For high-resolution immunofluorescence detection of At3g44130 in plant tissues:

Sample preparation:

• Fix tissue samples in 4% paraformaldehyde in PBS (pH 7.4) for 30-60 minutes

• Permeabilize with either:

  • 0.1-0.5% Triton X-100 (for membrane proteins)

  • 1-2% NP-40 (for cytosolic proteins)

  • Methanol/acetone (for nuclear proteins)

• Block with 3% BSA or 5% normal serum in PBS with 0.1% Tween-20

Antibody application:

• Apply primary At3g44130 antibody at 1:100-1:500 dilution, incubate overnight at 4°C

• Wash 3-5 times with PBS-T (PBS with 0.1% Tween-20)

• Apply fluorophore-conjugated secondary antibody (1:200-1:1000), incubate 2 hours at RT

• Counterstain nuclei with DAPI (1 μg/ml) for 10 minutes

• Mount in anti-fade mounting medium

Advanced visualization techniques:

• Confocal microscopy: For subcellular localization in whole-mount tissues

• Super-resolution microscopy: For nano-scale localization patterns

• Multi-channel imaging: Co-stain with organelle markers for precise localization

• Live cell imaging: For temporal dynamics studies using fluorescent protein fusions

To distinguish plasma membrane from cytosolic localization, use protoplasts or plasmolysis experiments where the plasma membrane detaches from the cell wall.

How do recent advancements in antibody technology apply to At3g44130 research?

Recent technological advances have expanded possibilities for At3g44130 antibody applications:

Recombinant antibody technologies:
Researchers can now produce fully human or synthetic antibodies against At3g44130 through approaches such as:

• Phage display selections using synthetic human single-chain fragment variable (scFv) libraries

• B-cell immortalization for monoclonal antibody development

• Directed evolution of antibody fragments for enhanced specificity

Enhanced validation strategies:
Current antibody validation standards require multiple lines of evidence:

• Orthogonal validation (correlation with mRNA expression)

• Genetic validation (testing in knockout systems)

• Independent antibody validation (multiple antibodies targeting different epitopes)

• Recombinant expression approaches

• Capture mass spectrometry

Advanced analytical applications:

• Antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene assays: While primarily developed for therapeutic antibodies, similar reporter systems could be adapted to study At3g44130 signaling pathways

• Direct energy-based optimization approaches: Novel computational methods for antibody design that could improve At3g44130 antibody specificity and affinity

• Trispecific antibody engineering: Advanced antibody engineering creating multi-specific recognition could be adapted for research applications requiring simultaneous detection of At3g44130 and interacting partners

What experimental approaches are recommended when At3g44130 antibody data contradicts other experimental findings?

When antibody-based At3g44130 data conflicts with other experimental results, a systematic troubleshooting approach is essential:

Validation steps for resolving contradictions:

Comprehensive reconciliation strategy:

• Re-validate antibody specificity:

  • Test on knockout/knockdown lines

  • Perform peptide competition assays

  • Check for cross-reactivity with similar proteins

• Consider biological variables:

  • Post-transcriptional regulation can explain RNA-protein discrepancies

  • Post-translational modifications may affect epitope recognition

  • Protein stability and turnover rates

  • Tissue-specific or stress-induced expression changes

• Address methodological differences:

  • Sample preparation variations (extraction buffers, fixation methods)

  • Detection sensitivity differences between methods

  • Quantification approach variations

• Employ orthogonal techniques:

  • CRISPR-Cas9 tagging of endogenous At3g44130

  • Proximity labeling approaches (BioID, TurboID)

  • Alternative visualization methods (split GFP)

A particularly powerful approach for resolving contradictions is generating multiple lines of evidence using independent methods similar to enhanced validation criteria for antibodies .

What are the best practices for using At3g44130 antibodies in chromatin immunoprecipitation (ChIP) experiments?

For successful ChIP experiments targeting At3g44130 (if it functions as a DNA-binding protein or chromatin-associated factor):

Optimized ChIP protocol:

• Crosslinking:

  • Fix Arabidopsis seedlings with 1% formaldehyde for 10-15 minutes under vacuum

  • Quench with 125 mM glycine for 5 minutes

• Chromatin preparation:

  • Grind tissue in liquid nitrogen

  • Extract nuclei using extraction buffer with protease inhibitors

  • Sonicate to fragment chromatin (200-500 bp fragments)

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with Protein A/G beads

  • Incubate with At3g44130 antibody (2 μl per 500 μl solution)

  • Include controls:

    • IgG control (non-specific antibody)

    • Input chromatin (non-immunoprecipitated)

    • Known target regions (positive control)

• Washing and elution:

  • Perform stringent washes to remove non-specific binding

  • Elute protein-DNA complexes

  • Reverse crosslinks (65°C overnight)

  • Purify DNA using column-based methods

• Analysis:

  • qPCR for known or predicted target regions

  • ChIP-seq for genome-wide binding profile

Optimization considerations:

• Antibody amount (titrate to determine optimal concentration)

• Chromatin amount (typically 25-100 μg per reaction)

• Incubation time (4-16 hours at 4°C)

• Wash stringency (adjust salt concentration based on antibody specificity)

• Sonication conditions (optimize to achieve desired fragment size)

For genome-wide studies, ChIP-seq library preparation and sequencing should follow standard protocols, with appropriate peak calling and statistical analysis.

How can At3g44130 antibodies be applied to study plant stress responses?

At3g44130 antibodies can be valuable tools for investigating plant stress responses through multiple experimental approaches:

Quantitative protein expression analysis:

• Subject Arabidopsis plants to different stress conditions:

  • Biotic stress (pathogen infection, insect herbivory)

  • Abiotic stress (drought, salt, cold, heat)

• Collect tissue samples at multiple time points

• Perform Western blot analysis with At3g44130 antibodies

• Quantify protein expression changes relative to controls and normalization standards

Co-immunoprecipitation for stress-induced interactions:

• Extract proteins from stressed and control plants

• Immunoprecipitate At3g44130 using specific antibodies

• Analyze co-precipitating proteins by mass spectrometry

• Identify stress-specific interaction partners

Subcellular localization changes:

• Perform subcellular fractionation on stressed and control tissues

• Analyze At3g44130 distribution by Western blot

• Alternatively, use immunofluorescence microscopy to track localization changes

Modifications and regulatory events:

• Use phospho-specific antibodies (if available) to monitor stress-induced phosphorylation

• Analyze protein stability and turnover rates under stress conditions

• Examine associations with stress-responsive transcription factors

This approach has been successfully applied to study plant immunity proteins like BIK1 (Botrytis-induced kinase 1), where antibody-based detection revealed important insights into stress response mechanisms .

What are the best approaches for multiplexed detection of At3g44130 and related proteins?

For simultaneous detection of At3g44130 and functionally related proteins:

Multiplexed Western blot strategies:

• Sequential probing:

  • Strip and reprobe membranes with different antibodies

  • Use distinct primary antibodies from different host species

• Multiplex fluorescent Western blotting:

  • Use primary antibodies from different species

  • Apply species-specific secondary antibodies with different fluorophores

  • Detect using fluorescent imaging systems

Multiplex immunofluorescence microscopy:

• Primary antibody combinations:

  • Select antibodies raised in different host species

  • Apply simultaneously or sequentially depending on protocol

• Detection systems:

  • Use species-specific secondary antibodies with distinct fluorophores

  • Ensure minimal spectral overlap between fluorophores

  • Include appropriate controls for cross-reactivity

• Advanced approaches:

  • Tyramide signal amplification for weak signals

  • Spectral unmixing for closely overlapping fluorophores

  • Sequential imaging with antibody stripping/elution

Mass spectrometry-based multiplexing:

• Co-immunoprecipitation:

  • Use a cocktail of antibodies against At3g44130 and related proteins

  • Analyze precipitated complexes by MS

• Tandem mass tag (TMT) labeling:

  • Immunoprecipitate proteins from different conditions

  • Label with isobaric mass tags

  • Combine and analyze by LC-MS/MS for quantitative comparison

How can researchers address non-specific binding issues with At3g44130 antibodies?

Non-specific binding is a common challenge with plant protein antibodies. For At3g44130 antibodies, implement these strategies:

Prevention approaches:

Validation and troubleshooting approaches:

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Apply to Western blot or immunostaining

  • Specific signals should disappear

• Knockout/knockdown controls:

  • Test antibody on tissue from At3g44130 mutant lines

  • Signals present in mutant indicate non-specific binding

• Signal verification by multiple methods:

  • Compare Western blot results with immunofluorescence

  • Verify with tagged protein expression

• Alternative detection systems:

  • Try different secondary antibodies or detection methods

  • Consider direct labeling of primary antibodies

• Sample preparation optimization:

  • Adjust extraction buffers to reduce interfering compounds

  • Include additives to reduce non-specific interactions (e.g., 0.1-1% Triton X-100, 0.1% SDS)

What techniques are recommended for studying developmental regulation of At3g44130 using antibodies?

To investigate the developmental regulation of At3g44130 expression and function:

Developmental expression profiling:

• Collect tissues at different developmental stages:

  • Seedling (3, 5, 7, 10 days)

  • Vegetative growth (rosette leaves, stems)

  • Reproductive development (flowers at different stages, siliques)

  • Senescence (aging leaves)

• Perform Western blot analysis with At3g44130 antibodies

• Quantify expression relative to loading controls

• Create comprehensive expression maps across development

Tissue-specific expression analysis:

• Immunohistochemistry approach:

  • Fix tissues at different developmental stages

  • Section using appropriate methods (paraffin, cryo, or vibratome)

  • Perform immunostaining with At3g44130 antibodies

  • Counterstain with cell type-specific markers

• Tissue fractionation approach:

  • Isolate specific tissues or cell types

  • Extract proteins and analyze by Western blot

  • Compare expression across different tissue types

Developmental interactome studies:

• Perform co-immunoprecipitation with At3g44130 antibodies from different developmental stages

• Analyze interacting partners by mass spectrometry

• Identify stage-specific protein interactions

Protein modifications during development:

• Examine patterns of post-translational modifications

• Look for proteolytic processing events

• Monitor protein stability and turnover

Functional studies:

• Correlate expression/localization patterns with developmental phenotypes in mutant lines

• Perform tissue-specific complementation studies

• Analyze genetic interactions with known developmental regulators