At5g55131 Antibody

What is the At5g55131 gene in Arabidopsis thaliana and what protein does it encode?

At5g55131 is a gene locus in the Arabidopsis thaliana genome that encodes a protein involved in autophagy-related pathways. Based on sequence homology and functional studies, it appears to participate in stress response mechanisms similar to other autophagy-related genes such as ATG5. The protein encoded by At5g55131 contains conserved domains that make it recognizable by specifically developed antibodies when using appropriate immunological techniques. Understanding this protein's function requires reliable antibodies for detection in various experimental contexts, particularly when studying plant stress responses and autophagy pathways .

How is specificity validated for antibodies targeting At5g55131 or similar Arabidopsis proteins?

Antibody specificity validation for At5g55131 or similar Arabidopsis proteins requires multiple complementary approaches:

• Western blot analysis using wild-type and knockout/mutant tissue samples (essential control)

• Testing cross-reactivity with related proteins from different species (human, mouse, yeast)

• Detection of the expected molecular weight band (comparing to theoretical predictions)

• Peptide competition assays to confirm epitope specificity

For example, in ATG5 antibody validation, researchers demonstrated specificity by showing detection of specific bands at approximately 50-51 kDa across multiple cell lines (HeLa human cervical epithelial carcinoma, CH-1 mouse B cell lymphoma, and PC-12 rat adrenal pheochromocytoma) . Similar approaches should be employed for At5g55131 antibodies, with particular attention to using proper negative controls like corresponding mutant lines (e.g., T-DNA insertion lines) where the protein is not expressed.

What expression patterns would researchers expect to detect using At5g55131 antibodies?

Based on data from similar autophagy-related proteins in Arabidopsis, researchers would expect to detect varied expression patterns depending on tissue type and stress conditions. For instance, promoter-reporter studies of autophagy-related genes like DPH1 show activity in all examined tissues with elevated expression in meristematic regions of shoot and root tissues . For At5g55131, researchers should anticipate:

• Baseline expression in most plant tissues

• Upregulation during stress conditions (particularly osmotic, nutrient deprivation)

• Potential subcellular localization patterns consistent with autophagy components

• Developmental stage-specific expression profiles

When designing immunolocalization studies, researchers should include appropriate controls and compare antibody staining patterns with transcript data from public repositories to ensure consistency with expected expression domains.

How can At5g55131 antibodies be optimized for immunoprecipitation in plant tissue samples?

Optimizing At5g55131 antibodies for immunoprecipitation (IP) in plant tissues requires careful consideration of several factors:

• Buffer composition: Plant tissues contain unique compounds that can interfere with antibody-antigen interactions. Include 0.5-1% Triton X-100 and 150-300mM NaCl in extraction buffers to minimize non-specific interactions while maintaining specific binding.

• Cross-linking approach: For transient or weak protein-protein interactions, consider using formaldehyde (1-2%) or DSP (dithiobis(succinimidyl propionate)) cross-linking prior to extraction.

• Pre-clearing strategy: Plant extracts often contain components that bind non-specifically to beads. Pre-clear lysates with Protein A/G beads for 1-2 hours before adding your antibody.

• Antibody concentration optimization: Test different antibody-to-lysate ratios (typically 2-10 μg antibody per 1 mg total protein) to determine optimal conditions for At5g55131 IP.

• Negative controls: Always include IP experiments using the same concentration of non-immune IgG and, when possible, samples from At5g55131 mutant plants .

This optimization strategy parallels approaches used successfully for other plant proteins, such as the DPH1-GFP detection in Arabidopsis root tips using confocal microscopy, where careful sample preparation and antibody concentration optimization were critical for specific signal detection .

What are the challenges in detecting post-translational modifications of At5g55131 protein?

Detecting post-translational modifications (PTMs) of At5g55131 presents several technical challenges:

• Modification-specific antibodies: Generating antibodies that specifically recognize modified forms (phosphorylated, ubiquitinated, etc.) requires careful immunogen design and extensive validation.

• Low abundance: Modified forms often exist at significantly lower concentrations than the unmodified protein, necessitating enrichment strategies.

• Tissue-specific or condition-dependent modifications: PTMs may only occur in specific tissues or under particular stress conditions, requiring systematic sampling.

• Epitope masking: Some modifications may block antibody recognition of nearby epitopes, requiring multiple antibodies targeting different regions.

• Technical verification: Mass spectrometry analysis is essential to confirm antibody-detected modifications.

These challenges parallel those encountered when studying diphthamide modification of eEF2 in Arabidopsis, where researchers needed specialized antibodies that could distinguish between modified and unmodified forms of the protein . For At5g55131, researchers should consider developing modification-specific antibodies if particular PTMs are critical to protein function.

How do researchers quantitatively assess antibody binding affinity for plant targets like At5g55131?

Quantitative assessment of antibody binding affinity for plant targets involves several complementary approaches:

When applying these methods to plant antibodies like those against At5g55131, researchers must carefully purify the target protein or use synthetic peptides representing key epitopes. The plant cellular environment contains compounds that can interfere with binding measurements, so sample preparation is critical for accurate affinity determination .

For example, in developability assessments of antibodies, researchers have established correlations between biophysical properties and key assay endpoints that predict downstream process parameters . Similar approaches can be applied to plant antibodies to quantitatively assess their binding characteristics and optimize their performance in various applications.

How can researchers minimize cross-reactivity when using At5g55131 antibodies in multi-protein detection assays?

Minimizing cross-reactivity in multi-protein detection assays requires several strategic approaches:

• Sequential immunoblotting: Strip and reprobe membranes sequentially rather than using cocktails of antibodies, allowing clear attribution of signals to specific antibodies.

• Species-diverse primary antibodies: When detecting multiple proteins, use primary antibodies raised in different species (rabbit anti-At5g55131, mouse anti-ATG8, etc.) to enable species-specific secondary antibody detection.

• Epitope competition testing: Pre-incubate the antibody with excess purified antigen or immunizing peptide to confirm signal specificity.

• Fluorophore selection for multiplexing: When using fluorescent detection, carefully select fluorophores with minimal spectral overlap and include single-antibody controls.

• Validation in mutant backgrounds: Test antibodies in genetic backgrounds where the target protein is absent or altered, such as T-DNA insertion lines for At5g55131 .

This approach follows validation practices seen with ATG5 antibodies, where researchers confirmed specificity across multiple species and cell lines through western blot analysis with carefully controlled conditions .

What are the optimal fixation and extraction protocols for immunolocalization of At5g55131 in different plant tissues?

Optimal fixation and extraction protocols for At5g55131 immunolocalization vary by tissue type:

For root tissues:

• Fix in 4% paraformaldehyde, 0.1% Triton X-100 in PBS for 1 hour at room temperature

• Wash 3× in PBS

• Cell wall digestion with 1% cellulase, 0.5% macerozyme in PBS for 15 minutes

• Permeabilize with 0.5% Triton X-100 for 15 minutes

• Block with 3% BSA, 0.1% Tween-20 in PBS for 1 hour

For leaf tissues:

• Fix in 4% paraformaldehyde, 0.1% glutaraldehyde in PBS for 2 hours

• Wash 3× in PBS

• Stronger cell wall digestion: 2% cellulase, 1% macerozyme, 0.5% pectinase for 30 minutes

• Permeabilize with 1% Triton X-100 for 30 minutes

• Block with 5% BSA, 0.1% Tween-20 in PBS for 2 hours

For reproductive tissues:

• Fix in 4% paraformaldehyde in PBS overnight at 4°C

• Wash 3× in PBS

• Embed in LR White resin for thin sectioning

• Retrieve antigens with citrate buffer (pH 6.0) heat treatment

• Block with 5% normal goat serum, 3% BSA in PBS for 2 hours

These protocols draw on approaches used for localizing proteins in Arabidopsis root tips, where researchers successfully detected cytosolic localization of proteins such as DPH1-GFP using confocal microscopy . The key factors for successful immunolocalization are adequate fixation, appropriate cell wall digestion, and effective blocking of non-specific binding sites.

How should western blot conditions be optimized specifically for At5g55131 detection?

Optimizing western blot conditions for At5g55131 detection requires systematic optimization of multiple parameters:

• Protein extraction buffer: Include protease inhibitors and 1% Triton X-100 to solubilize membrane-associated proteins effectively.

• Sample preparation: Heat samples at 70°C instead of 95°C to prevent potential aggregation of transmembrane domains if present.

• Gel percentage optimization:

  • 10% acrylamide gel for optimal resolution around the expected molecular weight

  • Consider gradient gels (4-15%) if working with multiple proteins of varying sizes

• Transfer conditions:

  • Semi-dry transfer: 15V for 30 minutes

  • Wet transfer: 30V overnight at 4°C for larger proteins

  • PVDF membrane (0.45 μm) generally performs better than nitrocellulose for plant proteins

• Blocking conditions:

  • 5% non-fat dry milk in TBST for standard blocking

  • For phospho-specific detection, use 5% BSA in TBST

• Antibody dilution optimization:

  • Test dilution series (1:500 to 1:5000) to determine optimal signal-to-noise ratio

  • Incubate primary antibody overnight at 4°C

• Detection system:

  • Enhanced chemiluminescence (ECL) provides good sensitivity

  • Fluorescent secondary antibodies allow for multiplex detection and more quantitative analysis

This approach follows protocols used successfully for ATG5 detection, where researchers used reducing conditions and specific immunoblot buffer groups to detect bands at approximately 50 kDa across multiple species .

How can researchers use At5g55131 antibodies to study protein-protein interactions in autophagy pathways?

Researchers can employ several complementary approaches to study At5g55131 protein-protein interactions in autophagy pathways:

• Co-immunoprecipitation (Co-IP): Use At5g55131 antibodies to pull down the protein complex, followed by mass spectrometry or western blotting for suspected interaction partners. This approach requires careful optimization of extraction conditions to maintain native protein complexes.

• Proximity-dependent biotin identification (BioID): Generate At5g55131-BioID fusion proteins to biotinylate proximal proteins, which can then be purified and identified.

• Förster resonance energy transfer (FRET): Combine fluorescently tagged At5g55131 with antibody-based detection of interaction partners to measure protein proximity in vivo.

• Split-GFP complementation: Engineer split-GFP constructs for At5g55131 and potential interacting partners, using antibodies to confirm expression levels.

• Cross-linking immunoprecipitation (CLIP): Use formaldehyde cross-linking to capture transient interactions before immunoprecipitation with At5g55131 antibodies.

This strategy builds on approaches used to study autophagy in Arabidopsis, where researchers identified interaction networks involving ATG proteins through various immunoprecipitation techniques . When identifying protein complexes, it's essential to include appropriate controls, such as IgG-only immunoprecipitations and experiments in mutant backgrounds lacking the target protein.

What controls are essential when using At5g55131 antibodies in stress response studies?

When studying stress responses with At5g55131 antibodies, several controls are critical for proper data interpretation:

• Genetic controls:

  • Wild-type vs. At5g55131 knockout/knockdown plants

  • Complementation lines (At5g55131 mutant expressing the wild-type gene)

  • Overexpression lines to assess dose-dependent effects

• Treatment controls:

  • Untreated controls matched for developmental stage and growth conditions

  • Time-course samples to distinguish early vs. late responses

  • Concentration gradients for chemical treatments

  • Recovery samples after stress removal

• Antibody controls:

  • Non-immune IgG at matching concentration

  • Peptide competition assays to confirm specificity

  • Secondary antibody-only controls

  • Cross-reactivity assessment with related proteins

• Technical controls:

  • Loading controls (anti-actin, anti-tubulin) for normalization

  • Samples from known stress-responsive pathways (positive controls)

  • Multiple biological replicates (minimum n=3)

These controls parallel those used in studies of autophagy activation in Arabidopsis dph1 mutants, where researchers included appropriate controls such as wild-type comparisons and treatments with the TOR kinase inhibitor AZD-8055 as a positive control for autophagy induction .

How can researchers quantitatively assess At5g55131 protein levels across different experimental conditions?

Quantitative assessment of At5g55131 protein levels requires rigorous methodology:

• Western blot quantification:

  • Use digital imaging systems rather than film

  • Ensure samples are in the linear range of detection

  • Normalize to multiple loading controls (e.g., GAPDH, actin)

  • Include calibration samples with known quantities of recombinant protein

• Enzyme-linked immunosorbent assay (ELISA):

  • Develop sandwich ELISA with capture and detection antibodies

  • Generate standard curves using purified recombinant At5g55131

  • Validate extraction protocols to ensure complete protein recovery

• Mass spectrometry approaches:

  • Selected reaction monitoring (SRM) for absolute quantification

  • Label-free quantification with appropriate normalization

  • Isotope-labeled standard peptides for precise quantification

• Automated western platforms:

  • Simple Western systems for higher reproducibility

  • Consistent sample loading and standardized protocols

  • Digital data acquisition for more reliable quantification

When implementing these methods, researchers should test different extraction buffers to ensure complete solubilization of At5g55131, as seen in studies of other plant proteins where extraction conditions significantly affected detection efficiency . For example, techniques like those used for detecting human and rat ATG5 by Simple Western can be adapted for plant proteins, where lysates are loaded at standardized concentrations (e.g., 0.2 mg/mL) and specific bands are quantified in comparison to standard curves .

How can At5g55131 antibodies be used in single-cell proteomics of plant tissues?

Single-cell proteomics using At5g55131 antibodies represents an emerging frontier in plant biology research:

• Mass cytometry (CyTOF) adaptation for plants:

  • Conjugate At5g55131 antibodies with rare earth metals

  • Optimize protoplast preparation to maintain protein integrity

  • Develop multiplexed panels with other autophagy markers

  • Implement computational approaches for high-dimensional data analysis

• Microfluidic antibody capture:

  • Design microfluidic chambers for single protoplast capture

  • Integrate on-chip antibody-based protein detection

  • Combine with transcriptomic analysis for multi-omic profiling

• In situ PLA (Proximity Ligation Assay):

  • Use pairs of antibodies against At5g55131 and potential interactors

  • Achieve single-molecule resolution within intact tissue contexts

  • Quantify interaction events in different cell types

• Spatial proteomics approaches:

  • Apply At5g55131 antibodies to tissue sections for spatial mapping

  • Integrate with laser capture microdissection for regional analyses

  • Correlate with functional responses in specific cell types

This emerging field builds on approaches being developed for other plant proteins and will enable researchers to understand cell type-specific regulation of At5g55131 during development and stress responses, similar to how tissue-specific expression patterns have been studied for other autophagy-related genes using reporter constructs and antibody detection .

What are the considerations for developing phospho-specific antibodies for At5g55131?

Developing phospho-specific antibodies for At5g55131 requires careful consideration of several factors:

• Phosphorylation site prediction and validation:

  • Use phosphoproteomics data and computational prediction tools to identify likely phosphorylation sites

  • Confirm sites through mass spectrometry analysis

  • Consider evolutionary conservation of phosphorylation sites across species

• Peptide design strategy:

  • Generate phosphopeptides (10-15 amino acids) with the phosphorylated residue centrally positioned

  • Include a terminal cysteine for conjugation if not present naturally

  • Consider synthesizing both phosphorylated and non-phosphorylated peptides for screening and validation

• Immunization and screening approach:

  • Immunize rabbits with the phosphopeptide conjugated to KLH carrier protein

  • Screen antibody specificity using both phosphorylated and non-phosphorylated peptides

  • Perform sequential affinity purification:

    • First against the non-phosphorylated peptide (negative selection)

    • Then against the phosphopeptide (positive selection)

• Validation in plant systems:

  • Test antibody specificity using samples treated with lambda phosphatase

  • Compare wild-type samples with mutants in relevant kinase pathways

  • Validate with phosphomimetic and phospho-null mutant constructs

This approach follows methodologies used for developing other post-translational modification-specific antibodies, where careful validation ensures specific detection of the modified form of the protein .

What are the key considerations for reproducible western blot analysis using At5g55131 antibodies?

Ensuring reproducible western blot analysis with At5g55131 antibodies requires adherence to several critical best practices:

• Standardized sample preparation:

  • Harvest tissue at consistent developmental stages and times of day

  • Use standardized buffer-to-tissue ratios for extraction

  • Document and maintain consistent protein quantification methods

  • Store aliquoted samples at -80°C, avoiding repeated freeze-thaw cycles

• Consistent gel electrophoresis parameters:

  • Maintain uniform acrylamide percentage between experiments

  • Use consistent sample loading amounts (10-20 μg total protein)

  • Include molecular weight markers in every gel

  • Document running conditions (voltage, amperage, duration)

• Optimized transfer and immunodetection:

  • Standardize transfer protocols (time, buffer composition, temperature)

  • Use consistent blocking reagents and durations

  • Prepare antibody dilutions from concentrated stocks rather than diluting previous dilutions

  • Document exposure times and detection methods precisely

• Proper controls and normalization:

  • Include positive and negative controls in each blot

  • Use multiple housekeeping proteins for normalization

  • Consider using total protein staining (SYPRO Ruby, Ponceau S) as an alternative normalization method

  • Report raw data alongside normalized values

These practices parallel those used in western blot detection of ATG5, where researchers maintained consistent conditions across experiments, including specific buffer groups and standardized protein concentrations . Implementing these practices will significantly improve reproducibility and reliability of At5g55131 detection across different experiments and laboratories.

How should researchers integrate antibody-based detection with other approaches to study At5g55131 function?

A comprehensive approach to studying At5g55131 function requires integration of antibody-based detection with multiple complementary methods:

• Multi-omics integration:

  • Correlate protein levels (antibody detection) with transcript levels (RNA-seq)

  • Combine with metabolomic analyses to link protein function to metabolic outcomes

  • Integrate phosphoproteomics to identify regulatory mechanisms

• Genetic and phenotypic correlation:

  • Compare antibody-detected protein levels across genetic variants (natural accessions, CRISPR mutants)

  • Link protein abundance to phenotypic outcomes under various stress conditions

  • Use inducible expression systems to perform time-course studies

• Structure-function analysis:

  • Combine antibody epitope mapping with protein domain function studies

  • Use antibodies to assess conformational changes under different conditions

  • Develop domain-specific antibodies to study protein processing events

• Cellular and subcellular context:

  • Correlate immunolocalization with live-cell imaging of fluorescent fusion proteins

  • Combine with cell fractionation to track protein movement between compartments

  • Integrate with interaction studies to build functional protein networks

This integrated approach follows successful strategies used to study autophagy in Arabidopsis, where researchers combined antibody detection with genetic analysis, marker proteins (ATG8a-GFP), and pharmacological treatments to build a comprehensive understanding of autophagy regulation . By integrating multiple complementary approaches, researchers can develop a more complete understanding of At5g55131 function in plant biology.