At3g63000 Antibody

What is AT3G63000 and why is it important for plant research?

AT3G63000 (also known as NPL41 or NPL4-like protein 1) is a protein-coding gene in Arabidopsis thaliana that belongs to the NPL4 protein family . This protein is significant in plant research because:

• It contains the NPL4 domain (InterPro:IPR007717)

• It is expressed in 25 plant structures across 15 growth stages

• According to SUBAcon consensus data, it localizes predominantly to the cytosol

• It appears in sulfenome mining studies, suggesting a role in redox regulation

• It may participate in endoplasmic reticulum-mediated protein quality control pathways similar to the ERAD (Endoplasmic Reticulum-Associated Degradation) system

The protein contains 413 amino acids with a predicted molecular weight of approximately 46.5 kDa and an isoelectric point of 5.12 . Understanding AT3G63000 contributes to our knowledge of plant stress responses and protein quality control mechanisms.

How can I detect AT3G63000 protein expression in plant tissues?

Detection of AT3G63000 requires selecting appropriate antibody-based techniques:

• Western blotting: The most common approach for detecting AT3G63000 protein expression in plant tissue extracts. Based on antibody protocols for other plant proteins , use the following methodology:

  • Extract total protein using a buffer containing reducing agents

  • Separate proteins on 10-12% SDS-PAGE gels

  • Transfer to PVDF membranes using standard protocols

  • Block with 5% non-fat milk or BSA

  • Incubate with primary AT3G63000 antibody (typically at 1-5 μg/mL)

  • Detect using appropriate HRP-conjugated secondary antibodies

  • Visualize using chemiluminescence

• Immunohistochemistry (IHC): For tissue localization studies:

  • Fix plant tissues with paraformaldehyde

  • Embed in paraffin and section (5-10 μm)

  • Perform antigen retrieval using heat-induced epitope retrieval with basic buffer (pH 9.0)

  • Block with appropriate serum

  • Incubate with AT3G63000 antibody (10-15 μg/mL overnight at 4°C)

  • Detect using HRP-DAB staining system with hematoxylin counterstain

• Immunofluorescence: For subcellular localization:

  • Use a protocol similar to that described for pollen nuclei preparation

  • Apply primary AT3G63000 antibody followed by fluorophore-conjugated secondary antibodies

  • Co-stain with organelle markers to confirm cytosolic localization predicted by SUBAcon

What controls should I include when using AT3G63000 antibodies?

For rigorous experimental design, include these essential controls:

• Positive control:

  • Wild-type Arabidopsis tissues known to express AT3G63000

  • Recombinant AT3G63000 protein (if available)

• Negative controls:

  • T-DNA insertion lines SALK_108611 or SALK_117593, which likely have reduced or absent AT3G63000 expression

  • Primary antibody omission control

  • Isotype control (using non-specific IgG of the same species)

• Specificity controls:

  • Pre-absorption of antibody with recombinant AT3G63000 protein

  • Western blot showing single band at expected molecular weight (~46.5 kDa)

  • Comparison with protein expression patterns in published literature

What are the key considerations for selecting an AT3G63000 antibody?

When selecting an antibody against AT3G63000, researchers should consider:

• Antibody type:

  • Polyclonal antibodies offer higher sensitivity but potentially lower specificity

  • Monoclonal antibodies provide higher specificity but might recognize only a single epitope

  • For novel targets like AT3G63000, polyclonal antibodies are often the initial choice

• Host species:

  • Choose a host species different from your experimental system to avoid cross-reactivity

  • Common hosts include rabbit, sheep, goat, or mouse

• Immunogen design:

  • Full-length recombinant protein (Ile22-Leu413) would be ideal

  • Alternatively, unique peptide sequences from AT3G63000 can be used

  • Avoid regions with high homology to other NPL4-family proteins

• Validation data:

  • Request evidence of specificity via Western blot

  • Check for cross-reactivity testing

  • Review immunohistochemistry images if available

• Application compatibility:

  • Ensure the antibody is validated for your application (WB, IHC, IP, etc.)

  • Some antibodies work well for Western blot but poorly for immunohistochemistry

How can I validate a new AT3G63000 antibody?

A systematic validation approach includes:

• Western blot analysis:

  • Test on wild-type plant extracts versus knockout/knockdown lines

  • Expected band size: ~46.5 kDa

  • Test different tissue types to confirm expression patterns

• Immunoprecipitation followed by mass spectrometry:

  • Pull down AT3G63000 protein using the antibody

  • Confirm identity via mass spectrometry

  • Similar to approaches used in sulfenome mining studies

• Immunostaining patterns:

  • Compare antibody staining with known subcellular localization (cytosol)

  • Use co-localization with established markers

• Genetic knockdown validation:

  • Compare staining in wild-type versus T-DNA insertion lines

  • Use available lines like SALK_108611 or SALK_117593

• Recombinant protein testing:

  • Express recombinant AT3G63000 (similar to protocols used for AG-3 protein)

  • Confirm antibody recognition of the recombinant protein

How can I use AT3G63000 antibodies for protein interaction studies?

For investigating protein interactions involving AT3G63000:

• Co-immunoprecipitation (Co-IP):

  • Lyse plant tissues in appropriate buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitors)

  • Incubate lysate with AT3G63000 antibody

  • Capture antibody-protein complexes using Protein A/G beads

  • Wash extensively and elute

  • Analyze by Western blot or mass spectrometry

  • Look for known ERAD pathway components that might interact with AT3G63000

• Proximity labeling combined with immunoprecipitation:

  • Similar to protocols used for sulfenome mining

  • Express fusion proteins with biotin ligase (BioID) or peroxidase (APEX)

  • Perform proximity labeling followed by pulldown with AT3G63000 antibody

  • Identify interaction partners by mass spectrometry

• Immunofluorescence co-localization:

  • Co-stain cells with AT3G63000 antibody and antibodies against suspected interaction partners

  • Analyze using confocal microscopy

  • Quantify co-localization using Pearson's correlation coefficient

What approaches can I use to study AT3G63000's role in protein quality control?

Based on its potential involvement in ERAD-like pathways , consider:

• Stress response experiments:

  • Treat plants with ER stress inducers (tunicamycin, DTT)

  • Monitor AT3G63000 protein levels by Western blot

  • Compare with known ERAD components

  • Include SALK_108611 or SALK_117593 mutant lines as controls

• Ubiquitylation analysis:

  • Immunoprecipitate with AT3G63000 antibody

  • Probe Western blots with anti-ubiquitin antibodies

  • Alternatively, perform tandem ubiquitin binding entity (TUBE) pulldowns

  • Compare ubiquitylation patterns between wild-type and stress conditions

• Proteasome inhibition studies:

  • Treat plants with MG132 (proteasome inhibitor)

  • Examine changes in AT3G63000 protein levels and interacting partners

  • Look for accumulated ERAD substrates

• Cycloheximide chase assays:

  • Treat plant cells with cycloheximide to inhibit protein synthesis

  • Monitor AT3G63000 protein degradation over time by Western blot

  • Compare degradation rates under normal and stress conditions

How can AT3G63000 antibodies be used in redox biology research?

Given AT3G63000's identification in sulfenome mining studies :

• Detection of oxidative modifications:

  • Perform dimedone labeling to trap sulfenic acids

  • Immunoprecipitate with AT3G63000 antibody

  • Detect oxidized forms using dimedone-specific antibodies

  • Similar to protocols described for DHAR2 sulfenylation analysis

• Redox proteomics workflow:

  • Treat plants with oxidative stress inducers (H₂O₂, paraquat)

  • Immunoprecipitate AT3G63000 using specific antibodies

  • Analyze post-translational modifications by mass spectrometry

  • Compare redox states using isotope-coded affinity tags

• Monitoring redox-dependent subcellular localization:

  • Perform immunofluorescence under normal and oxidative stress conditions

  • Quantify changes in subcellular distribution

  • Co-stain with organelle markers

How might redox regulation affect AT3G63000's function in protein quality control?

Based on AT3G63000's presence in sulfenome datasets and potential ERAD involvement :

• Mechanistic hypothesis:

  • AT3G63000 contains 5 cysteine residues that may be susceptible to oxidation

  • Oxidative stress could modify these cysteines, potentially altering protein function

  • These modifications might regulate AT3G63000's interactions with ERAD machinery

• Experimental approach:

  • Site-directed mutagenesis of cysteine residues

  • Expression of mutant proteins in SALK T-DNA backgrounds

  • Analysis of protein quality control function under normal and stress conditions

  • Monitoring of protein-protein interactions with ERAD components

• Advanced analytical methods:

  • Hydrogen-deuterium exchange mass spectrometry to analyze conformational changes

  • NMR studies of oxidized versus reduced protein

  • Proximity ligation assays to detect stress-dependent interactions in situ

What is the relationship between AT3G63000 and other NPL4-family proteins in plants?

For comparative studies of NPL4-family proteins:

• Phylogenetic analysis:

  • AT3G63000 (NPL41) is most similar to AT2G47970.1 (Nuclear pore localisation protein NPL4)

  • Compare functional domains and evolutionary conservation

  • Analyze expression patterns across different tissues and conditions

• Functional redundancy testing:

  • Generate single and double knockout lines using CRISPR/Cas9

  • Compare phenotypes of single mutants versus double mutants

  • Use AT3G63000-specific antibodies to confirm protein absence

• Cross-reactivity assessment:

  • Test AT3G63000 antibodies against recombinant NPL4-family proteins

  • Perform epitope mapping to identify unique regions

  • Develop antibodies against unique epitopes if cross-reactivity occurs

• Transcript-protein correlation analysis:

  • Compare mRNA expression (via RT-qPCR) with protein levels (via antibody detection)

  • Analyze across different tissues and stress conditions

  • Identify post-transcriptional regulation mechanisms

How does AT3G63000 function in the context of plant stress responses?

To investigate AT3G63000's role in stress adaptation:

• Stress response profiling:

  • Expose plants to various stresses (drought, salt, heat, pathogen)

  • Monitor AT3G63000 protein levels and post-translational modifications

  • Compare with known stress response markers

  • Analyze phenotypes of SALK_108611 or SALK_117593 mutants under stress

• Protein quality control assessment:

  • Measure accumulation of ubiquitylated proteins in wild-type versus mutant plants

  • Monitor ER stress markers (e.g., BiP, PDI)

  • Analyze unfolded protein response activation

  • Quantify protein aggregation under stress conditions

• Systems biology approach:

  • Perform RNA-Seq and proteomics on wild-type versus mutant plants

  • Construct protein-protein interaction networks

  • Identify pathways affected by AT3G63000 deletion

  • Validate key interactions using co-immunoprecipitation with AT3G63000 antibodies

What should I do if I encounter non-specific binding with my AT3G63000 antibody?

To resolve non-specific binding issues:

• Antibody optimization:

  • Titrate antibody concentration (try 0.1-5 μg/mL range)

  • Extend blocking time (2-3 hours or overnight)

  • Try different blocking agents (BSA, non-fat milk, normal serum)

  • Increase washing steps and duration

• Sample preparation improvements:

  • Use fresher tissue samples

  • Include additional protease inhibitors

  • Add reducing agents to prevent disulfide cross-linking

  • Pre-clear lysates with Protein A/G beads

• Specificity enhancement:

  • Pre-absorb antibody with Arabidopsis lysate from knockout lines

  • Increase salt concentration in washing buffers (up to 500 mM NaCl)

  • Add 0.1-0.5% SDS or 0.1-1% Triton X-100 to washing buffers

  • Use more stringent washing conditions for immunoprecipitation

• Alternative detection methods:

  • Try a different secondary antibody

  • Use direct conjugation of primary antibody

  • Consider signal amplification systems for weak signals

How can I optimize immunofluorescence staining for AT3G63000?

For improved immunofluorescence results:

• Fixation optimization:

  • Compare different fixatives (4% paraformaldehyde, methanol, acetone)

  • Adjust fixation time (10-30 minutes)

  • Try different permeabilization methods (0.1-0.5% Triton X-100, 0.1% saponin)

• Antigen retrieval:

  • Test heat-induced epitope retrieval methods (similar to protocols for AG-3)

  • Try different pH conditions (citrate buffer pH 6.0 vs. Tris-EDTA pH 9.0)

  • Optimize retrieval time (10-30 minutes)

• Signal enhancement:

  • Use tyramide signal amplification

  • Try different fluorophore-conjugated secondary antibodies

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

  • Reduce autofluorescence with sodium borohydride treatment

• Advanced imaging:

  • Apply deconvolution algorithms

  • Use super-resolution microscopy for improved localization

  • Perform quantitative co-localization analysis

How can I determine if my AT3G63000 antibody is recognizing the correct protein?

To validate antibody specificity:

• Genetic validation:

  • Compare signal between wild-type and SALK T-DNA insertion lines

  • Perform complementation with tagged AT3G63000 and co-detection

  • Use CRISPR/Cas9-generated knockout lines as definitive controls

• Biochemical confirmation:

  • Perform mass spectrometry on immunoprecipitated protein

  • Compare observed molecular weight with predicted size (46.5 kDa)

  • Check for expected post-translational modifications

• Cross-validation:

  • Compare results from multiple antibodies targeting different epitopes

  • Use alternative detection methods (e.g., expressing tagged protein)

  • Correlate protein levels with mRNA expression data

• Competitive binding assay:

  • Pre-incubate antibody with recombinant AT3G63000 protein

  • Observe signal reduction in Western blot or immunofluorescence

  • Establish concentration-dependent inhibition curve