At2g12475 Antibody

What is the At2g12475 protein and why is it studied in Arabidopsis thaliana?

At2g12475 is a protein encoded by the At2g12475 gene in Arabidopsis thaliana (Mouse-ear cress), with UniProt accession number Q2V488. This protein is studied primarily in plant molecular biology research as part of understanding gene expression and protein function in this model organism. While specific functions of this protein are still being elucidated, antibodies against it serve as important tools for tracking its expression, localization, and interactions within plant cells .

To begin studying this protein, researchers typically combine genomic data with proteomic approaches, using antibodies as key reagents for detection and characterization. The antibody enables visualization of the protein's distribution in different tissues and subcellular compartments, which helps establish its biological role.

What applications are suitable for At2g12475 antibody in basic research?

At2g12475 antibody can be used in several standard immunological techniques:

• Western blotting for protein detection and semi-quantification

• Immunohistochemistry (IHC) for localization in fixed tissue samples

• Immunocytochemistry (ICC) for subcellular localization

• ELISA for quantitative measurement

• Immunoprecipitation for studying protein interactions

For beginners, Western blotting represents the most accessible starting point, as it provides clear information about protein expression and apparent molecular weight. When designing experiments, use positive and negative controls to validate antibody specificity .

How should I store and handle At2g12475 antibody to maintain its activity?

For optimal performance and longevity of the At2g12475 antibody:

• Store concentrated antibody at -20°C when not in use

• For working solutions, store at 2-8°C for short periods (typically 1-2 weeks)

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Do not expose to strong light for extended periods

• Maintain sterile conditions when handling

• Check the expiration date provided by the manufacturer

For long-term storage, adding stabilizing proteins such as BSA (0.1-1%) can help preserve antibody activity. When diluting the antibody, use buffers recommended by the manufacturer, typically PBS with 0.1% BSA and 0.05% sodium azide .

How can I validate the specificity of At2g12475 antibody in Arabidopsis tissues?

Validation of At2g12475 antibody specificity is crucial for reliable research outcomes. Implement these methodological approaches:

• Genetic validation: Test the antibody on wild-type versus knockout/knockdown plants lacking At2g12475 expression. The signal should be absent or significantly reduced in the knockout samples.

• Blocking peptide experiments: Pre-incubate the antibody with excess purified At2g12475 protein or immunizing peptide before application. Specific signals should be abolished.

• Multiple antibody approach: Use two different antibodies raised against different epitopes of At2g12475. Concordant results strengthen confidence in specificity.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein.

• Cross-reactivity testing: Test the antibody against related plant species to determine evolutionary conservation and specificity.

Document all validation experiments methodically with appropriate controls to establish antibody reliability for your specific experimental conditions .

What are the considerations for using At2g12475 antibody in co-localization studies with other plant proteins?

For successful co-localization studies involving At2g12475:

• Antibody compatibility: Ensure the At2g12475 antibody (CSB-PA648071XA01DOA) can be used alongside antibodies against other proteins of interest by checking host species to avoid cross-reactivity.

• Spectral separation: When using fluorescent secondary antibodies, select fluorophores with minimal spectral overlap to prevent false co-localization signals.

• Fixation optimization: Different fixation protocols may variably preserve epitopes. Test multiple fixation methods (paraformaldehyde, glutaraldehyde, methanol) to determine optimal conditions for all target proteins.

• Sequential staining protocol:

  • Apply primary antibody against At2g12475

  • Wash thoroughly (minimum 3×10 minutes)

  • Apply second primary antibody

  • Wash thoroughly

  • Apply appropriate secondary antibodies with distinct fluorophores

  • Include appropriate controls (single antibody staining, secondary-only controls)

• Advanced imaging considerations: Use confocal microscopy with sequential scanning rather than simultaneous acquisition to minimize bleed-through, and perform proper controls for co-localization analysis, such as Pearson's correlation coefficient calculation .

How can At2g12475 antibody be used in studying plant stress responses?

To investigate At2g12475's role in plant stress responses:

• Experimental design for stress studies:

  • Expose Arabidopsis plants to various stressors (drought, salt, temperature, pathogens)

  • Collect tissue samples at multiple time points

  • Process for protein extraction and analysis using the At2g12475 antibody

• Quantitative approaches:

  • Western blot with densitometry analysis

  • ELISA for precise quantification

  • Immunohistochemistry with quantitative image analysis

• Subcellular redistribution analysis:

  • Use cell fractionation followed by Western blotting

  • Perform immunofluorescence microscopy before and after stress application

  • Analyze changes in localization patterns

• Protein modification detection:

  • Use 2D-gel electrophoresis followed by Western blotting to identify post-translational modifications

  • Combine with phospho-specific antibodies if phosphorylation is suspected

• Protein interaction dynamics:

  • Perform co-immunoprecipitation under different stress conditions

  • Analyze changes in interaction partners using mass spectrometry

Document all experimental conditions meticulously, including stress parameters, tissue types, and protein extraction methods, as these factors significantly influence results .

What is the optimal protocol for using At2g12475 antibody in Western blotting of plant proteins?

For optimal Western blotting with At2g12475 antibody:

Sample preparation and electrophoresis:

• Extract total protein from Arabidopsis tissues using a plant-specific extraction buffer containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Load 20-50 µg of protein per lane (optimize based on expression level)

• Separate proteins using SDS-PAGE (10-12% gel recommended for most plant proteins)

Transfer and immunoblotting:

• Transfer proteins to PVDF or nitrocellulose membrane (PVDF often provides better results for plant proteins)

• Block membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hour at room temperature

• Incubate with At2g12475 antibody (CSB-PA648071XA01DOA) at 1:1000 dilution (optimize as needed) overnight at 4°C

• Wash 3 times with TBST, 10 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Wash 3 times with TBST, 10 minutes each

• Develop using ECL substrate and document using chemiluminescence imaging system

Optimization considerations:

• Test different antibody dilutions (1:500 to 1:5000)

• Vary blocking agents if background is high

• Adjust incubation times and temperatures

• Consider using gradient gels if protein size is uncertain

For plant proteins, include controls for non-specific binding, which is sometimes more prevalent than with animal proteins .

How should I perform immunohistochemistry with At2g12475 antibody on plant tissue sections?

For successful immunohistochemistry with At2g12475 antibody on plant tissues:

Tissue preparation:

• Fix fresh plant tissues in 4% paraformaldehyde in PBS for 12-24 hours at 4°C

• Dehydrate through ethanol series (30%, 50%, 70%, 95%, 100%)

• Clear in xylene and embed in paraffin

• Section at 5-10 µm thickness and mount on positively charged slides

Immunostaining protocol:

• Deparaffinize sections with xylene and rehydrate through descending ethanol series

• Perform antigen retrieval (citrate buffer pH 6.0, 95°C for 20 minutes)

• Block endogenous peroxidase with 3% H₂O₂ if using HRP detection

• Block non-specific binding with 5% normal serum from the species of the secondary antibody

• Apply At2g12475 antibody (1:100 to 1:500 dilution, optimize) and incubate overnight at 4°C

• Wash 3 times with PBS, 5 minutes each

• Apply biotinylated secondary antibody for 1 hour at room temperature

• Wash 3 times with PBS, 5 minutes each

• Apply streptavidin-HRP or other detection system

• Develop with DAB or fluorescent reagents

• Counterstain, dehydrate, clear, and mount as appropriate

Plant-specific considerations:

• Cell wall interference may require increased permeabilization

• Autofluorescence from chlorophyll and other plant compounds necessitates appropriate controls and quenching steps

• Include negative controls (primary antibody omission, non-immune serum substitution)

• Include positive controls (tissues known to express the target) .

What controls should I include when performing immunoprecipitation with At2g12475 antibody?

When performing immunoprecipitation with At2g12475 antibody, include these essential controls:

Input control:

• Save 5-10% of the pre-cleared lysate before adding the antibody

• Run this sample alongside IP samples to confirm the presence of target protein in starting material

Negative controls:

• IgG control: Perform parallel IP with normal IgG from the same species as the At2g12475 antibody

• Null tissue control: Use tissue from knockout/knockdown plants lacking At2g12475

• Beads-only control: Process a sample without antibody to identify proteins binding non-specifically to beads

Specificity controls:

• Peptide competition: Pre-incubate antibody with excess immunizing peptide before IP

• Reverse IP: If studying protein interactions, confirm by immunoprecipitating with antibodies against suspected interaction partners

Methodology validation:

• Test various lysis buffers as plant proteins may require different solubilization conditions

• Include protease and phosphatase inhibitors appropriate for plant tissues

• Optimize antibody-to-lysate ratios (typically 2-5 μg antibody per mg of total protein)

Data interpretation table:

Document all experimental conditions, including buffer compositions, incubation times, and washing stringency, as these significantly affect results .

Why might I be getting weak or no signal when using At2g12475 antibody in Western blotting?

If experiencing weak or absent signals with At2g12475 antibody, systematically troubleshoot using this methodology:

• Protein extraction issues:

  • Ensure sufficient protein is loaded (30-50 μg for most plant proteins)

  • Verify extraction buffer compatibility with plant tissues

  • Add protease inhibitors to prevent degradation

  • Verify protein transfer by staining membrane with Ponceau S

• Antibody-related factors:

  • Check antibody concentration (try more concentrated solutions: 1:500 instead of 1:1000)

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

  • Ensure antibody storage conditions have been appropriate

  • Verify the antibody hasn't expired

• Epitope accessibility:

  • Try different membrane types (PVDF often works better than nitrocellulose for plant proteins)

  • Reduce methanol concentration in transfer buffer for high MW proteins

  • Test different antigen retrieval methods (heat, SDS, etc.)

  • Consider native vs. reducing conditions (some epitopes are destroyed by reducing agents)

• Detection system:

  • Switch to more sensitive detection methods (enhanced ECL substrates)

  • Check secondary antibody compatibility and functionality

  • Increase exposure time during imaging

  • Use fresh detection reagents

• Protein expression factors:

  • Verify protein expression in your specific tissues/conditions

  • Consider developmental timing or stress conditions that might affect expression

  • Use positive control samples with known expression

Methodological approach for optimization:
Test variables systematically, changing only one parameter at a time and documenting results to determine the optimal protocol for your specific experimental conditions .

How can I reduce background when using At2g12475 antibody in immunofluorescence of plant tissues?

To reduce background in immunofluorescence with At2g12475 antibody:

• Optimized blocking strategy:

  • Increase blocking agent concentration (5-10% serum or BSA)

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

  • Include 0.1-0.3% Triton X-100 in blocking buffer for better penetration

  • Try different blocking agents (milk, BSA, normal serum, fish gelatin)

  • Add 0.1% glycine to quench free aldehyde groups from fixation

• Plant-specific autofluorescence reduction:

  • Pretreat sections with 0.1% Sudan Black B in 70% ethanol

  • Use 0.1M NH₄Cl to reduce fixative-induced autofluorescence

  • Include 0.1% NaBH₄ treatment step

  • Use confocal microscopy settings to minimize chlorophyll autofluorescence

• Antibody optimization:

  • Further dilute primary antibody (test 1:200, 1:500, 1:1000)

  • Reduce incubation temperature to 4°C

  • Add 0.05% Tween-20 to antibody dilution buffer

  • Pre-absorb antibody with plant tissue powder from unrelated species

• Washing optimization:

  • Increase number and duration of washes (5×10 minutes)

  • Use PBS-T with higher detergent concentration (0.1-0.3% Triton X-100)

  • Include salt washes (PBS with 0.5M NaCl) to reduce ionic interactions

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Further dilute secondary antibody

  • Centrifuge secondary antibody before use to remove aggregates

  • Switch to fragment antibodies (Fab) if necessary

Systematic optimization approach:
Create a grid experiment testing combinations of blocking agents and washing conditions to identify optimal parameters for your specific tissue type .

What strategies can I use to increase specificity in co-immunoprecipitation experiments with At2g12475 antibody?

To enhance specificity in co-immunoprecipitation experiments with At2g12475 antibody:

• Buffer optimization:

  • Adjust salt concentration (150-500 mM NaCl) to reduce non-specific interactions

  • Test different detergents (NP-40, Triton X-100, CHAPS) at varying concentrations

  • Include competitors for non-specific interactions (0.1-0.5% BSA)

  • Add specific components for plant protein stabilization (glycerol, specific ions)

• Pre-clearing optimization:

  • Extend pre-clearing step with beads alone (1-2 hours)

  • Use both protein A and protein G beads for comprehensive pre-clearing

  • Include non-immune IgG in pre-clearing step

  • Pre-absorb lysate with plant tissue powder

• Antibody-specific strategies:

  • Cross-link antibody to beads to prevent antibody leaching

  • Test different antibody amounts (2-10 μg per sample)

  • Optimize antibody incubation time and temperature

  • Consider using magnetic beads instead of agarose for cleaner results

• Washing optimization:

  • Design a stringency gradient for washes (start with milder, end with more stringent)

  • Increase number of washes (5-7 washes)

  • Include detergent in all wash buffers

  • Perform the final 1-2 washes with detergent-free buffer

• Technical refinements:

  • Use low-binding tubes to reduce non-specific protein loss

  • Keep samples consistently cold throughout the procedure

  • Minimize handling time to reduce protein degradation

  • Consider formaldehyde cross-linking to stabilize weak or transient interactions

Validation of interaction specificity:

Document all parameters methodically to establish reproducible conditions for specific interaction detection .

How can I quantitatively analyze Western blot data for At2g12475 protein expression across different experimental conditions?

For rigorous quantitative analysis of At2g12475 expression across conditions:

• Proper experimental design:

  • Include technical replicates (minimum 3)

  • Process all samples simultaneously when possible

  • Include appropriate loading controls (plant housekeeping proteins like actin or tubulin)

  • Use a standard curve with known protein amounts if absolute quantification is needed

• Image acquisition considerations:

  • Ensure signals are within linear detection range (not saturated)

  • Use identical exposure settings for all blots being compared

  • Capture images in an uncompressed format (TIFF preferred over JPG)

  • Use the same imaging system for all blots in a comparative study

• Densitometry methodology:

  • Use software designed for Western blot analysis (ImageJ, Image Lab, etc.)

  • Define identical regions of interest (ROIs) for all bands

  • Subtract local background individually for each lane

  • Normalize target protein signal to loading control

  • Express results as relative to control condition

• Statistical analysis:

  • Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple)

  • Test data for normality before choosing parametric/non-parametric tests

  • Report mean ± standard deviation (or SEM) with n value clearly stated

  • Include p-values and significance thresholds

Data presentation format example:

Present data graphically with appropriate error bars, clearly indicating statistical significance .

How can I interpret contradictory results between Western blot and immunohistochemistry when using At2g12475 antibody?

When facing contradictory results between Western blot (WB) and immunohistochemistry (IHC) using At2g12475 antibody:

• Understand fundamental differences between techniques:

  • WB detects denatured proteins; IHC detects proteins in more native conformation

  • WB provides information on protein size; IHC provides spatial information

  • WB is typically more quantitative; IHC offers localization insights

• Methodological causes for discrepancies:

  Potential Issue	Western Blot Consideration	IHC Consideration	Resolution Approach
  Epitope accessibility	Denaturation exposes epitopes	Fixation may mask epitopes	Try different fixation methods for IHC; use native PAGE for WB
  Cross-reactivity	Size separation helps distinguish targets	Spatial context can be misleading	Perform peptide competition controls in both methods
  Sensitivity threshold	Concentrated samples detect low abundance	Localized concentrations may be below detection	Use more sensitive detection for IHC; concentrate samples for WB
  Post-translational modifications	May alter antibody recognition	Could affect epitope exposure	Use phospho-specific antibodies if modification is suspected
  Fixation artifacts	N/A	Aldehyde fixation can create false epitopes	Use multiple fixation protocols in IHC

• Comprehensive validation strategy:

  • Perform both techniques on the same tissue preparation when possible

  • Include genetic controls (knockout/knockdown plants)

  • Use multiple antibodies targeting different epitopes

  • Complement with mRNA expression data (RT-qPCR, in situ hybridization)

  • Consider fluorescence correlation spectroscopy for quantitative in situ validation

• Biological interpretation of discrepancies:

  • Protein may exist in different conformational states in different cellular compartments

  • Processing or degradation products may be detected differently by each method

  • Aggregation or complex formation may mask epitopes in one method but not the other

  • Differential expression across cell types may be averaged in WB but visible in IHC

Use contradictory results as an opportunity to discover novel biological insights about protein processing, localization, or modification states .

How can At2g12475 antibody be used in studying protein-protein interactions in Arabidopsis thaliana?

For investigating protein-protein interactions involving At2g12475:

• Co-immunoprecipitation (Co-IP) methodology:

  • Optimize lysis conditions for plant tissues (test multiple buffers)

  • Use mild detergents (0.5-1% NP-40 or Triton X-100) to preserve complexes

  • Include stabilizing agents (5-10% glycerol)

  • Perform IP with At2g12475 antibody and blot for suspected interaction partners

  • Confirm with reverse Co-IP using antibodies against interacting proteins

  • Validate with recombinant proteins if available

• Proximity ligation assay (PLA) for in situ detection:

  • Requires At2g12475 antibody and antibody against suspected interactor from different host species

  • Provides direct visualization of interactions in fixed plant tissues

  • Optimize fixation to preserve both epitopes

  • Include controls: single antibodies, non-interacting protein pairs

  • Quantify signal dots per cell to assess interaction strength under different conditions

• Bimolecular Fluorescence Complementation (BiFC) complementary approach:

  • While not using the antibody directly, can validate interactions identified by Co-IP

  • Clone At2g12475 and interaction partner into split fluorescent protein vectors

  • Express in plant protoplasts or via transient transformation

  • Visualize fluorescence restoration when proteins interact

  • Use antibody in parallel experiments to confirm expression levels

• Co-localization combined with FRET:

  • Use At2g12475 antibody with differently labeled secondary antibody

  • Use antibody against potential interaction partner with complementary fluorophore

  • First confirm co-localization by confocal microscopy

  • Perform FRET analysis to determine proximity (<10 nm)

  • Analyze FRET efficiency under different biological conditions

• Mass spectrometry validation workflow:

  • Perform IP with At2g12475 antibody

  • Analyze by LC-MS/MS to identify all co-precipitating proteins

  • Filter against appropriate controls (IgG IP, beads only)

  • Validate top candidates by targeted approaches (Co-IP, PLA)

  • Create interaction network using bioinformatics tools

Data integration strategy:
Combine multiple interaction detection methods to build confidence in true interactions. Create an interaction score based on detection across multiple methodologies .

How can At2g12475 antibody be used in chromatin immunoprecipitation (ChIP) experiments if the protein has DNA-binding properties?

For using At2g12475 antibody in ChIP experiments:

• ChIP protocol optimization for plant tissues:

  • Crosslink fresh plant tissue with 1% formaldehyde (10-15 minutes)

  • Quench with glycine (125 mM final concentration)

  • Isolate nuclei using plant-specific isolation buffers (containing protease inhibitors)

  • Sonicate chromatin to 200-500 bp fragments (optimize cycles empirically)

  • Pre-clear chromatin with protein A/G beads

  • Immunoprecipitate with At2g12475 antibody (typically 2-5 μg per reaction)

  • Include IgG control and input samples

  • Reverse crosslinks (65°C overnight)

  • Purify DNA for downstream analysis

• Critical control experiments:

  • Input DNA (non-immunoprecipitated) control

  • IgG negative control

  • Positive control (antibody against known chromatin-associated protein)

  • ChIP from tissue with At2g12475 knockout/knockdown

  • ChIP-seq control (spike-in normalization recommended)

• Downstream analysis options:

  • ChIP-qPCR for targeted regions

  • ChIP-seq for genome-wide binding profile

  • Re-ChIP to analyze co-occupancy with other factors

  • ChIP-mass spectrometry to identify protein partners at chromatin

• Plant-specific ChIP considerations:

  • Cell wall interference requires optimization of tissue grinding

  • Polysaccharide contamination may require additional purification steps

  • High background can be reduced with more stringent washing

  • Formaldehyde penetration may be limited by waxy cuticles

• Data validation approaches:

  • Motif analysis of binding sites

  • Correlation with gene expression data

  • Comparison with published ChIP-seq datasets

  • Functional analysis of bound genes (GO term enrichment)

ChIP-seq data analysis workflow:

Document all experimental variables including fixation time, sonication parameters, antibody concentrations, and washing conditions .

Can At2g12475 antibody be used for super-resolution microscopy techniques, and what special considerations apply?

For applying At2g12475 antibody in super-resolution microscopy:

• Compatibility with different super-resolution techniques:

  Technique	Compatibility Factors	Special Considerations
  STED (Stimulated Emission Depletion)	Requires photostable fluorophores	Use secondary antibodies with STED-optimized dyes (Abberior Star, Atto 647N)
  STORM/PALM (Stochastic Optical Reconstruction)	Requires photoswitchable fluorophores	Conjugate with Alexa Fluor 647 or similar; optimize switching buffer
  SIM (Structured Illumination)	Less demanding on fluorophores	Standard fluorophores acceptable; high signal-to-noise ratio critical
  Expansion Microscopy	Antibody must withstand gelation process	Test antibody retention after expansion; may require re-staining

• Sample preparation optimization:

  • Use thinner sections (≤5 μm) for better resolution

  • Optimize fixation carefully (over-fixation reduces antibody penetration)

  • Consider tissue clearing techniques (ClearSee, CLARITY adapted for plants)

  • Use smaller fluorophore conjugates when possible

  • For plant tissues, cell wall digestion may improve antibody accessibility

• Labeling strategies for improved resolution:

  • Consider directly labeled primary antibodies to reduce linkage error

  • Use Fab fragments instead of full IgG to decrease distance to target

  • For STORM/PALM, optimize labeling density (too high causes overlap)

  • For dual-color imaging, ensure minimal chromatic aberration through channel alignment

• Plant-specific adaptations:

  • Manage autofluorescence with appropriate filters or spectral unmixing

  • Use cell wall counterstains compatible with super-resolution (calcofluor white for STED)

  • Account for refractive index changes at cell wall/membrane interfaces

  • Consider the 3D nature of plant cells requiring Z-stacking

• Controls and validation:

  • Include single-label controls for multicolor imaging

  • Validate with conventional microscopy first

  • Use known subcellular markers for co-localization studies

  • Test specificity with competition assays adapted to super-resolution

The theoretical resolution achievable depends on the technique: STED/STORM/PALM can reach 20-50 nm lateral resolution, while SIM typically achieves 100-120 nm resolution, all significantly better than the diffraction limit (~250 nm) .

How can At2g12475 antibody contribute to understanding plant protein degradation pathways, particularly related to autophagy?

To investigate At2g12475's potential role in plant protein degradation and autophagy:

• Experimental approaches to track protein degradation:

  • Perform cycloheximide chase assays with At2g12475 antibody detection

    • Treat plants with cycloheximide to block protein synthesis

    • Collect samples at time intervals (0, 2, 4, 8, 24 hours)

    • Western blot with At2g12475 antibody to track degradation kinetics

    • Calculate protein half-life

  • Inhibitor studies to identify degradation pathways

    • MG132 (proteasome inhibitor)

    • E-64d, leupeptin (lysosomal/vacuolar protease inhibitors)

    • 3-methyladenine, wortmannin (autophagy inhibitors)

    • Compare At2g12475 levels by Western blot after inhibitor treatments

• Co-localization with autophagy markers:

  • Immunofluorescence using At2g12475 antibody alongside antibodies against:

    • ATG8 (key autophagy marker, similar to LC3 in mammals)

    • ATG5-ATG12 complex (important for autophagosome formation)

    • Vacuolar markers (tonoplast proteins)

  • Induction conditions to test:

    • Nutrient starvation (nitrogen or carbon limitation)

    • Oxidative stress (H₂O₂ treatment)

    • Development-specific stages

• Integration with the ATG12-ATG5 conjugation system:

  • Co-immunoprecipitation with At2g12475 antibody followed by blotting for:

    • ATG12 (part of the E3-like enzyme complex in autophagy)

    • ATG5 (conjugated partner of ATG12)

    • ATG16L1 (forms complex with ATG5-ATG12)

  • Test whether At2g12475 is modified by ATG12 system (higher MW band detection)

  • Investigate if At2g12475 is recruited to autophagosomes under stress

• Examination of selective autophagy pathways:

  • If At2g12475 is selectively degraded, test co-localization with:

    • NBR1/Joka2 (plant selective autophagy receptors)

    • Ubiquitin (possible degradation signal)

    • Organelle-specific markers (mitochondria, chloroplasts, ER) to identify potential mitophagy, chlorophagy, or ER-phagy

• Quantitative analysis methodology:

  • Measure co-localization coefficients (Pearson's, Mander's) between At2g12475 and autophagy markers

  • Quantify changes in At2g12475 protein levels under autophagy-inducing conditions

  • Track co-localization changes over time during autophagy induction

  • Measure autophagic flux using At2g12475 as a potential cargo

Understanding the relationship between At2g12475 and the autophagy machinery would provide insights into plant-specific adaptations of this conserved degradation pathway, potentially revealing novel regulatory mechanisms .

How does the specificity of At2g12475 antibody compare to antibodies against homologous proteins in other plant species?

For comparing specificity across plant species homologs:

• Cross-reactivity assessment methodology:

  • Perform Western blot analysis with At2g12475 antibody on protein extracts from:

    • Arabidopsis thaliana (source species)

    • Close relatives (other Brassicaceae: Brassica, Capsella)

    • More distant dicots (tomato, tobacco, soybean)

    • Monocots (rice, maize, wheat)

    • Non-vascular plants (moss, liverwort) if relevant

  • Document band patterns, molecular weights, and signal intensities

  • Confirm identity of cross-reactive bands by mass spectrometry

• Epitope conservation analysis:

  • Identify the epitope used to generate the At2g12475 antibody

  • Perform sequence alignment of homologous proteins across species

  • Calculate percent identity and similarity in the epitope region

  • Correlate sequence conservation with observed cross-reactivity

• Functional domain mapping:

  • Test whether antibody recognition corresponds to conserved functional domains

  • Compare recognition patterns with protein domain predictions

  • Use domain-specific predictions to explain partial cross-reactivity

• Prediction of cross-reactivity:

  Plant Species	Epitope Homology	Predicted Cross-reactivity	Empirical Results
  Arabidopsis thaliana	100% (reference)	High (control)	Strong single band
  Brassica species	85-95%	Moderate to high	To be determined
  Solanum species	60-75%	Low to moderate	To be determined
  Oryza sativa	40-55%	Very low	To be determined
  Physcomitrella patens	30-45%	Negligible	To be determined

• Applications of cross-reactivity information:

  • Use conserved epitope antibodies for evolutionary studies

  • Develop species-specific antibodies for regions with low conservation

  • Leverage cross-reactivity for comparative studies of protein function

  • Document species limitations for accurate experimental design

This systematic approach allows researchers to determine whether At2g12475 antibody can serve as a tool for studying homologous proteins across plant lineages, expanding its utility beyond Arabidopsis research .

What considerations apply when using At2g12475 antibody in combination with other antibodies for multiplex detection systems?

For successful multiplex detection with At2g12475 antibody:

• Antibody compatibility assessment:

  • Host species considerations:

    • Ensure primary antibodies are from different host species (e.g., rabbit anti-At2g12475 with mouse anti-other target)

    • Alternatively, use directly conjugated primaries to avoid host conflicts

    • Consider isotype differences if antibodies are from same species

  • Cross-reactivity testing:

    • Test each antibody individually first

    • Perform sequential staining with second primary omitted as control

    • Test for secondary antibody cross-reactivity independently

• Spectral compatibility for fluorescence:

  • Fluorophore selection strategy:

    • Choose fluorophores with minimal spectral overlap

    • Account for plant autofluorescence when selecting channels

    • Consider brightness differences (balance exposure settings)

    • For super-resolution techniques, ensure all fluorophores are compatible with the method

  • Recommended fluorophore combinations:

    • For three-color: AF488 (green), AF568 (red), AF647 (far-red)

    • For four-color: DAPI (blue), AF488 (green), AF568 (red), AF647 (far-red)

    • For plant tissues with chlorophyll: avoid using GFP channel, prefer far-red dyes

• Multiplexed Western blot strategies:

  • Sequential detection methods:

    • Strip and reprobe (document complete stripping)

    • Use spectrally distinct fluorescent secondaries

    • Employ different detection chemistries (e.g., chemiluminescence + chromogenic)

  • Molecular weight considerations:

    • Ensure targets have sufficiently different sizes

    • Use loading controls distant from targets of interest

    • Consider using different membrane regions for overlapping proteins

• Quantitative considerations in multiplex detection:

  • Signal normalization approaches:

    • Use consistent control samples across experiments

    • Account for differential antibody affinities

    • Establish standard curves for each target if absolute quantification is needed

  • Avoiding detection interference:

    • Test for antigen-dependent antibody blocking effects

    • Validate that signal from one channel doesn't affect others

    • Use appropriate compensation controls for flow cytometry

• Advanced multiplexing technologies:

  • Mass cytometry (CyTOF) for high-plex detection:

    • Conjugate At2g12475 antibody with specific metal isotopes

    • Allows for 30+ parameter detection without spectral overlap

    • Requires specialized equipment but eliminates autofluorescence issues

  • Multiplexed immunohistochemistry:

    • Cyclic immunofluorescence with repeated staining/stripping

    • Multiplexed ion beam imaging (MIBI)

    • Imaging mass cytometry for tissue sections

Document all optimization steps and validation controls to ensure reliable multiplex detection without artifacts or false interpretations .

How does At2g12475 antibody perform in different detection systems (chemiluminescence vs. fluorescence vs. chromogenic) for Western blotting?

Comprehensive comparison of detection systems for At2g12475 antibody in Western blotting:

• Chemiluminescence detection:

  • Sensitivity profile:

    • Detection threshold: typically 10-100 pg of target protein

    • Dynamic range: 2-3 orders of magnitude (enhanced chemiluminescence)

    • Signal duration: minutes to hours depending on substrate

  • Optimization parameters:

    • Substrate choice (standard ECL vs. enhanced ECL)

    • Exposure time optimization (30 seconds to 10 minutes)

    • Film vs. digital imaging considerations

    • HRP-conjugated secondary antibody dilution (typically 1:5000-1:20000)

  • Advantages for At2g12475 detection:

    • High sensitivity for low abundance plant proteins

    • Compatible with membrane stripping and reprobing

    • No special equipment beyond standard darkroom/imager required

  • Limitations:

    • Signal can saturate, limiting quantitative accuracy

    • Temporal decay of signal introduces variability

    • Single target detection per experiment unless stripped

• Fluorescence detection:

  • Sensitivity profile:

    • Detection threshold: typically 1-10 ng (standard), 100 pg (enhanced)

    • Dynamic range: 3-4 orders of magnitude

    • Signal stability: days to weeks when protected from light

  • Optimization parameters:

    • Fluorophore selection (Alexa Fluor, IRDye, DyLight series)

    • Scanner/imager settings (laser power, gain)

    • Membrane autofluorescence considerations (PVDF vs. nitrocellulose)

    • Secondary antibody concentration (typically 1:10000-1:20000)

  • Advantages for At2g12475 detection:

    • Superior multiplexing capability (2-3 targets simultaneously)

    • Better linearity for quantification

    • No substrate limitations or signal decay

  • Limitations:

    • Higher initial equipment cost (fluorescence scanners)

    • Potential plant pigment interference with certain fluorophores

    • Less sensitive than optimal chemiluminescence systems for some applications

• Chromogenic detection:

  • Sensitivity profile:

    • Detection threshold: typically 50-100 ng

    • Dynamic range: 1-2 orders of magnitude

    • Signal stability: months to years when properly stored

  • Optimization parameters:

    • Substrate choice (DAB, NBT/BCIP, TMB)

    • Development time (3-30 minutes typically)

    • Enhancer addition for increased sensitivity

    • Secondary antibody concentration (typically 1:1000-1:5000)

  • Advantages for At2g12475 detection:

    • Permanent record without signal decay

    • Visual monitoring of development

    • No specialized equipment needed

    • Minimal background with optimized blocking

  • Limitations:

    • Lowest sensitivity of all methods

    • Limited quantitative capacity

    • Difficult to strip and reprobe

Comparative performance matrix: