At1g32140 Antibody

What is the At1g32140 antibody and what protein does it target in Arabidopsis thaliana?

At1g32140 antibody is a polyclonal antibody developed against proteins encoded by the At1g32140 gene locus in Arabidopsis thaliana. Similar to other plant antibodies like anti-At1g32060, these antibodies are typically generated using synthetic peptides derived from specific amino acid sequences of the target protein . The antibody recognizes protein products from the At1g32140 locus and can be used in various immunological applications including western blot and ELISA. Comparable antibodies in this class are often produced in rabbit hosts and purified using antigen affinity methods to ensure specificity .

What are the recommended storage conditions for At1g32140 antibody?

Based on similar plant antibodies, At1g32140 antibody should be stored at -20°C to -80°C to maintain activity and prevent degradation . For lyophilized formats, reconstitution with sterile water is typically recommended before use, and once reconstituted, the antibody should be divided into small aliquots to avoid repeated freeze-thaw cycles which can compromise antibody performance . Always remember to briefly centrifuge antibody tubes prior to opening to collect any material that might adhere to the cap or sides of the tube .

What is the typical working dilution range for At1g32140 antibody in western blot applications?

For western blot applications, a typical working dilution range for plant antibodies like At1g32140 is 1:1000 to 1:5000 . As with similar Arabidopsis thaliana antibodies, optimal dilution may vary depending on sample concentration, detection method, and specific experimental conditions . It is recommended to perform preliminary experiments with different dilutions to determine the optimal concentration for your specific application and experimental setup.

How should I validate the specificity of At1g32140 antibody before use in critical experiments?

Validating antibody specificity is crucial for reliable experimental results. For At1g32140 antibody, consider implementing the following validation strategy:

• Positive and negative controls: Include wild-type Arabidopsis samples (positive control) and knockout/knockdown lines of At1g32140 (negative control).

• Pre-absorption test: Pre-incubate the antibody with the immunizing peptide before immunodetection. If the antibody is specific, the signal should be significantly reduced or eliminated.

• Cross-reactivity assessment: Test the antibody against recombinant proteins with similar sequences to evaluate potential cross-reactivity.

• Multiple detection methods: Confirm results using at least two independent techniques (e.g., western blot and immunofluorescence) .

These validation steps help ensure experimental reliability and reproducibility when using At1g32140 antibody in research applications.

What protein extraction methods are most effective when working with At1g32140 antibody for western blot analysis of Arabidopsis samples?

Effective protein extraction is critical for successful western blot analysis with At1g32140 antibody. The following extraction protocol is recommended based on protocols used with similar plant antibodies:

Recommended Extraction Protocol:

• Grind 100 mg of plant tissue in liquid nitrogen to a fine powder

• Add 500 μl of extraction buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Vortex thoroughly and incubate on ice for 30 minutes with occasional mixing

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect the supernatant and determine protein concentration

For membrane-associated proteins, consider adding 0.5% sodium deoxycholate to the extraction buffer to improve solubilization. When extracting from chloroplast-rich tissues, specialized buffers may be required to effectively isolate proteins from these organelles, similar to approaches used for other chloroplastic proteins in Arabidopsis .

How can I optimize immunoprecipitation protocols using At1g32140 antibody?

Optimizing immunoprecipitation (IP) with At1g32140 antibody requires careful consideration of buffer conditions and antibody-to-lysate ratios. Based on protocols for similar plant antibodies:

• Pre-clearing: Incubate lysate with protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Antibody binding: Use 2-5 μg of At1g32140 antibody per 500 μg of total protein lysate. Incubate overnight at 4°C with gentle rotation.

• Buffer optimization: Test different buffer conditions:

  Buffer Component	Standard Condition	Stringent Condition	Mild Condition
  NaCl	150 mM	300 mM	100 mM
  Detergent	0.1% Triton X-100	0.5% Triton X-100	0.05% Triton X-100
  pH	7.4	8.0	7.0
  Wash steps	3 × 5 min	5 × 5 min	2 × 5 min

• Cross-linking: Consider using DSS or BS3 cross-linker to covalently link the antibody to protein A/G beads to prevent antibody co-elution.

• Elution method: Compare acidic elution (0.1 M glycine, pH 2.5) with SDS-PAGE sample buffer elution to determine which provides better recovery while minimizing background.

Optimizing these parameters will help maximize target protein recovery while minimizing non-specific interactions.

What are common causes of high background when using At1g32140 antibody in western blots?

High background is a frequent challenge when working with plant antibodies. For At1g32140 antibody, consider these potential causes and solutions:

Some researchers report success with adding 0.05% Tween-20 to antibody dilution buffers and using TBS instead of PBS for plant protein applications .

How can I use At1g32140 antibody to investigate protein-protein interactions in chloroplast biology?

At1g32140 antibody can be a valuable tool for studying protein-protein interactions in chloroplast biology using these advanced approaches:

• Co-immunoprecipitation (Co-IP): Use At1g32140 antibody to pull down its target protein along with interacting partners. Analyze the immunoprecipitated complex by mass spectrometry to identify novel interactions.

• Proximity-dependent biotin identification (BioID): Fuse a biotin ligase to your protein of interest, then use At1g32140 antibody to confirm expression and localization before streptavidin pulldown of biotinylated proximal proteins.

• Bimolecular Fluorescence Complementation (BiFC): Validate interactions identified through Co-IP using BiFC, where At1g32140 antibody can serve as a control for expression levels.

• Sucrose gradient fractionation: Use At1g32140 antibody to detect the target protein in different fractions to identify multiprotein complexes.

When examining interactions in chloroplast biology, consider using protocols specifically designed for chloroplast isolation to maintain structural integrity, similar to approaches used for other chloroplastic proteins in Arabidopsis .

How should I interpret discrepancies between At1g32140 antibody results and transcriptomic data?

Discrepancies between protein levels (detected by At1g32140 antibody) and transcript levels may result from several biological factors:

• Post-transcriptional regulation: mRNA stability and translation efficiency can significantly impact protein levels without changes in transcript abundance. Measure mRNA half-life using actinomycin D treatment followed by qRT-PCR.

• Post-translational modifications: Modifications may affect antibody recognition without altering transcript levels. Consider using phosphatase treatment or mass spectrometry to identify modifications.

• Protein turnover rates: Different degradation rates can lead to discrepancies. Use cycloheximide chase assays to determine protein half-life.

• Compartmentalization: Proteins may be sequestered in different cellular compartments. Perform cell fractionation followed by western blotting to determine localization patterns.

• Technical limitations: Antibody affinity and specificity can influence detection sensitivity. Validate results using multiple antibodies or tagged protein constructs.

A comprehensive approach to resolving such discrepancies would include correlation analysis between protein and transcript levels across multiple timepoints or treatments, which can reveal regulatory patterns and mechanisms.

How can At1g32140 antibody be used in chromatin immunoprecipitation (ChIP) experiments for studying protein-DNA interactions?

While many plant antibodies are not specifically validated for ChIP, At1g32140 antibody may be adapted for this application with careful optimization. Consider the following protocol adaptations:

• Crosslinking optimization: Test different formaldehyde concentrations (0.75%-3%) and incubation times (5-20 minutes) to optimize crosslinking while preserving epitope accessibility.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp, as larger fragments may increase background signal.

• Antibody amount: ChIP typically requires more antibody than western blot. Start with 5-10 μg antibody per reaction and adjust based on results.

• Controls: Always include:

  • Input DNA (non-immunoprecipitated chromatin)

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

  • Positive control (antibody against histone modifications)

• Washing stringency: Use a series of increasingly stringent wash buffers to reduce non-specific binding:

  • Low salt buffer (150 mM NaCl)

  • High salt buffer (500 mM NaCl)

  • LiCl buffer (250 mM LiCl)

  • TE buffer

Verify ChIP efficiency using qPCR before proceeding to next-generation sequencing to ensure sufficient enrichment of target regions.

What are the considerations for using At1g32140 antibody in mass spectrometry-based proteomics studies?

When using At1g32140 antibody for immunoprecipitation prior to mass spectrometry analysis, consider these critical factors:

• Antibody purity: Contaminants in the antibody preparation can interfere with mass spectrometry analysis. Use highly purified antibody preparations and include appropriate controls.

• Cross-linking considerations: If using cross-linking to stabilize protein-protein interactions:

  • Select MS-compatible cross-linkers (e.g., DSS, BS3, or DSSO)

  • Optimize cross-linker concentration and reaction time

  • Consider using cleavable cross-linkers for improved peptide identification

• Sample preparation:

  • Avoid detergents incompatible with MS (e.g., SDS)

  • Consider filter-aided sample preparation (FASP) or SP3 methods

  • Use MS-grade trypsin for protein digestion

• Data analysis workflow:

  Analysis Step	Recommended Tools	Considerations
  Peptide identification	MaxQuant, PEAKS	Use appropriate false discovery rate controls
  Protein quantification	MaxQuant LFQ, Skyline	Account for missing values
  Interaction filtering	CRAPome, SAINT	Filter against appropriate controls
  Network analysis	String-DB, Cytoscape	Integrate with known interactome data

• Validation: Confirm key interactions using orthogonal methods such as co-immunoprecipitation followed by western blot.

These considerations help ensure reliable results when using At1g32140 antibody for proteomics applications in plant research.

How can I use At1g32140 antibody to investigate protein localization changes during plant stress responses?

At1g32140 antibody can be valuable for studying dynamic changes in protein localization during stress responses using these approaches:

• Subcellular fractionation: Separate cellular compartments (chloroplast, mitochondria, cytosol, nucleus) from control and stressed plants, then use western blotting with At1g32140 antibody to track protein redistribution.

• Immunofluorescence microscopy: Perform immunostaining with At1g32140 antibody on fixed Arabidopsis tissues or protoplasts under different stress conditions.

• Time-course experiments: Analyze samples at multiple timepoints during stress exposure to capture dynamic localization changes.

• Co-localization studies: Combine At1g32140 antibody with markers for specific organelles to confirm localization patterns.

Experimental design considerations:

For all experiments, include appropriate controls for antibody specificity and consider using transgenic lines expressing fluorescently tagged versions of the protein as complementary approaches.

How does At1g32140 antibody specificity compare to antibodies against related Arabidopsis proteins?

When comparing At1g32140 antibody to antibodies against related Arabidopsis proteins, researchers should consider these key factors:

• Epitope selection: Similar to anti-At1g32060 antibody, which targets a specific peptide sequence (amino acids 223-235) , At1g32140 antibody specificity depends heavily on the immunizing peptide selection. Regions with high sequence homology to related proteins can lead to cross-reactivity.

• Cross-reactivity profile: Based on analysis of similar antibodies:

  Antibody	Primary Target	Potential Cross-Reactivity	Distinguishing Features
  At1g32140	At1g32140 protein	Closely related homologs	Specific to unique epitope regions
  At1g32060	Phosphoribulokinase	Similar chloroplastic proteins	Recognizes AA 223-235 region
  GOX1,2,3	Glycolate oxidase	Multiple GOX isoforms	Detects all three GOX isoforms

• Application-specific performance: Antibodies that perform well in western blot may not be optimal for immunoprecipitation or immunofluorescence. Always validate for each specific application.

• Recommended controls: When evaluating specificity, include:

  • Recombinant target protein

  • Tissue from knockout/knockdown plants

  • Pre-absorption with immunizing peptide

  • Comparative analysis with antibodies targeting different epitopes of the same protein

Understanding these factors helps researchers select the most appropriate antibody for their specific experimental needs.

What are the key methodological differences when using At1g32140 antibody compared to antibodies against human proteins?

Working with plant antibodies like At1g32140 requires different methodological approaches compared to antibodies against human proteins:

• Extraction buffer composition: Plant tissues contain unique compounds that can interfere with antibody-antigen interactions:

  • Include polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds

  • Add higher concentrations of reducing agents (5-10 mM DTT) to manage oxidative enzymes

  • Consider plant-specific protease inhibitor cocktails that target different protease classes

• Membrane blocking: Plant extracts may cause different background patterns:

  • Test different blocking agents (milk vs. BSA vs. casein)

  • Consider longer blocking times (2 hours to overnight)

  • Evaluate plant-specific blocking reagents that address unique non-specific interactions

• Antibody incubation conditions:

  • Plant antibodies often require lower temperatures (4°C) and longer incubation times

  • Higher salt concentrations in wash buffers may be needed to reduce background

• Detection systems:

  • Plant autofluorescence can interfere with immunofluorescence techniques

  • Endogenous plant peroxidases may cause background in HRP-based detection systems

• Sample preparation for microscopy:

  • Plant cell walls require different fixation and permeabilization methods

  • Consider enzymatic digestion of cell walls prior to antibody incubation

These methodological differences highlight the importance of optimizing protocols specifically for plant-based immunological applications.