At2g41170 Antibody

What is At2g41170 and what is its biological function in Arabidopsis thaliana?

At2g41170 is a gene encoding a protein in Arabidopsis thaliana with UniProt accession number Q6NKN8 . Based on available data, the protein is significant enough to warrant antibody development for research purposes. While the specific biological function is not explicitly detailed in the search results, researchers can investigate this protein's role by utilizing the antibody in various experimental contexts. The gene appears to be expressed under the CaMV 35S promoter in research constructs, suggesting it may have regulatory functions that researchers are actively studying .

For comprehensive functional characterization, researchers should consider combining antibody-based detection with genetic approaches such as gene knockout studies or overexpression systems. The availability of a plasmid containing AT2G41170 decoy recombined into the pK7-HFN destination vector (with N-terminal His-FLAG tags) provides additional tools for functional studies .

What applications can the At2g41170 antibody be used for?

The At2g41170 antibody has been validated for several key molecular biology applications:

• Western blot (WB): For detecting the native and denatured protein in tissue or cell lysates

• Enzyme-linked immunosorbent assay (ELISA): For quantitative detection in solution-based assays

The antibody is antigen-affinity purified, which enhances its specificity for the target protein . For Western blot applications, researchers should consider optimizing protein extraction methods specifically for plant tissues, which often contain compounds that can interfere with immunodetection. Based on protocols for similar plant antibodies, a recommended starting dilution would be 1:1000 to 1:3000, with overnight incubation at 4°C .

What are the recommended storage and handling conditions for At2g41170 antibody?

For optimal performance and stability of the At2g41170 antibody, follow these storage and handling recommendations:

• Upon receipt, store the antibody at -20°C or -80°C

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• The antibody is supplied in liquid form with the following buffer composition:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 (preservative)

When working with the antibody, always centrifuge tubes briefly before opening to ensure all material is collected at the bottom, similar to protocols used for other plant antibodies . For long-term storage stability, consider making small aliquots to minimize freeze-thaw cycles. Based on standard antibody handling practices, thawed aliquots should be kept at 4°C if they will be used within 1-2 weeks.

How should I optimize Western blot protocols for At2g41170 antibody?

Based on protocols used for similar plant antibodies, consider the following optimization strategies:

• Sample Preparation:

  • Extract proteins using a buffer containing protease inhibitors to prevent degradation

  • For microsomal fractions (if targeting membrane-associated proteins), follow protocols similar to those described for other plant proteins

  • Determine optimal protein loading (5-10μg has been effective for similar antibodies)

• Blocking Conditions:

  • Try 5% milk in PBS-T (PBS with 0.1% Tween 20) for 1 hour at room temperature

  • Alternative: 1% BSA in TBS-T may reduce background for some applications

• Antibody Incubation:

  • Primary antibody: Start with 1:1000 to 1:3000 dilution, incubate overnight at 4°C

  • Secondary antibody: Anti-rabbit IgG-HRP at 1:10,000 to 1:20,000 dilution for 1-2 hours at room temperature

• Detection:

  • ECL-based chemiluminescence with exposure times of 5-6 minutes has worked well with similar plant antibodies

When optimizing, prepare a dilution series of both sample and antibody to determine the best signal-to-noise ratio for your specific experimental conditions.

What controls should I include when working with At2g41170 antibody?

For rigorous experimental design, include the following controls:

• Positive Control:

  • Recombinant At2g41170 protein or overexpression system in Arabidopsis

  • Tissues known to express At2g41170 at detectable levels

• Negative Controls:

  • Knockout or knockdown lines lacking At2g41170 expression

  • Pre-immune serum at the same concentration as the primary antibody

  • Secondary antibody only (omitting primary antibody)

• Loading Controls:

  • Housekeeping proteins such as actin, tubulin, or GAPDH

  • Total protein staining methods (Ponceau S, Coomassie, or Stain-Free technology)

• Specificity Controls:

  • Peptide competition assay using the immunizing peptide to confirm binding specificity

  • Comparison with orthogonal detection methods (e.g., mass spectrometry)

These controls will help validate experimental findings and address potential reviewers' concerns regarding antibody specificity and experimental rigor.

What approaches can I use to validate the specificity of At2g41170 antibody?

Comprehensive validation of antibody specificity is critical for publication-quality research. Consider these approaches:

• Genetic Validation:

  • Compare wild-type vs. knockout/knockdown lines

  • Use CRISPR-Cas9 edited lines with specific mutations in the At2g41170 gene

  • Test overexpression lines with tagged versions of At2g41170

• Biochemical Validation:

  • Peptide competition assays using the immunizing peptide/protein

  • Mass spectrometry analysis of immunoprecipitated proteins

  • Immunoprecipitation followed by western blotting with a different antibody targeting another epitope of At2g41170

• Cross-Reactivity Testing:

  • Test the antibody against closely related proteins from the same family

  • Assess reactivity in heterologous expression systems

• Epitope Mapping:

  • Determine the specific region recognized by the antibody using truncated protein fragments

  • This information can help predict potential cross-reactivity with related proteins

What are best practices for using At2g41170 antibody in co-immunoprecipitation studies?

For successful co-immunoprecipitation (Co-IP) studies with At2g41170 antibody:

• Buffer Optimization:

  • Test multiple lysis/extraction buffers with varying ionic strengths and detergents

  • Include protease and phosphatase inhibitors to preserve protein-protein interactions

  • Consider crosslinking reagents for transient or weak interactions

• Antibody Coupling:

  • Directly couple the antibody to beads (protein A/G or magnetic) to avoid IgG contamination

  • Use proper controls including:

    • Pre-immune serum or IgG from the same species

    • Unrelated antibody of the same isotype

    • Input sample (pre-IP material)

• Enrichment Strategies:

  • For low-abundance proteins, consider using the PK7HFN-AT2G41170 construct that contains N-terminal His-FLAG tags

  • This allows tandem purification approaches that can increase specificity

• Interaction Verification:

  • Confirm interactions bidirectionally (IP with anti-At2g41170 and with antibodies against interacting partners)

  • Validate interactions using orthogonal methods (yeast two-hybrid, FRET, BiFC)

• Analysis Methods:

  • Mass spectrometry for unbiased identification of interacting partners

  • Western blotting for targeted detection of suspected interaction partners

These approaches will help identify and validate true interacting partners of At2g41170 while minimizing false positives.

How can I optimize protein extraction methods for At2g41170 detection in plant tissues?

Effective protein extraction is crucial for reliable At2g41170 detection. Consider these specialized approaches:

• Tissue-Specific Considerations:

  • For Arabidopsis, approximately 1g of plant material is recommended for sufficient protein yield

  • Flash-freeze harvested tissue in liquid nitrogen and store at -80°C until extraction

  • Grind tissues thoroughly while keeping samples frozen (using mortar and pestle or tissue lyser)

• Extraction Buffer Optimization:

  • For total protein: Use buffers containing 50mM Tris-HCl (pH 8.0) with appropriate detergents

  • For microsomal fractions: Follow established protocols for membrane protein enrichment

  • Include protease inhibitors, reducing agents, and EDTA to preserve protein integrity

• Fractionation Approaches:

  • Consider subcellular fractionation if At2g41170 has known or predicted compartmentalization

  • Sequential extraction methods can help identify the protein's association with different cellular components

• Quantification and Quality Control:

  • Determine protein concentration using Bradford or BCA methods

  • Assess extract quality with Coomassie staining before immunoblotting

  • Verify protein integrity with housekeeping protein detection

• Special Treatments:

  • For phosphorylation studies, include phosphatase inhibitors in all buffers

  • For studying protein responses to stimuli, consider appropriate treatment conditions (e.g., flg22 infiltration for 10 minutes has been used for similar proteins)

These optimized extraction methods will maximize the chances of detecting At2g41170 reliably across different experimental conditions.

What challenges might I encounter when detecting low-abundance At2g41170 in plant samples?

Detecting low-abundance proteins in plant tissues presents several challenges:

• Plant-Specific Interference:

  • Phenolic compounds, polysaccharides, and secondary metabolites can interfere with protein extraction and detection

  • Solution: Include PVPP, β-mercaptoethanol, and/or PVP in extraction buffers

• Signal Enhancement Strategies:

  • Super-sensitive ECL substrates for chemiluminescence detection

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

  • Signal amplification systems (tyramide signal amplification or catalyzed reporter deposition)

  • Consider using the tagged version of At2g41170 in the pK7-HFN vector which allows for His-FLAG tag detection

• Protein Enrichment Approaches:

  • Immunoprecipitation before western blotting

  • Subcellular fractionation to concentrate the target protein

  • Protein concentration methods (TCA precipitation, acetone precipitation)

• Technical Optimization:

  • Higher antibody concentration (1:500 dilution)

  • Extended exposure times during imaging

  • Increased protein loading (15-30μg may be necessary)

  • Transfer optimization (longer transfer times or specialized buffers)

• Alternative Detection Methods:

  • Consider multiple antibody applications (ELISA may be more sensitive than western blotting for some applications)

  • Mass spectrometry-based targeted proteomics (SRM/MRM)

By addressing these challenges systematically, researchers can improve detection of low-abundance At2g41170 even in complex plant samples.

How can I assess antibody performance in different experimental conditions?

Systematic evaluation of antibody performance across various conditions is essential for robust research:

For each parameter, document the results systematically to create a reliable protocol for future experiments. This approach will help establish reproducible conditions for detecting At2g41170 across different experimental systems and between laboratories.

What is known about the post-translational modifications of At2g41170?

While the search results do not provide specific information about post-translational modifications (PTMs) of At2g41170, researchers can design experiments to investigate potential modifications using the available antibody:

• Experimental Approaches to Detect PTMs:

  • Phosphorylation: Use Phos-tag gels or phospho-specific antibodies if available

  • Ubiquitination: Immunoprecipitate with At2g41170 antibody followed by ubiquitin detection

  • Glycosylation: Use glycosidase treatments and observe mobility shifts

  • SUMOylation: Look for higher molecular weight bands under specific conditions

• Induction of PTMs:

  • Test various stress conditions (e.g., pathogen exposure, abiotic stress)

  • Hormone treatments relevant to plant defense signaling

  • Developmental timing and tissue-specific expression

• PTM Analysis Tools:

  • Mass spectrometry for unbiased PTM identification

  • Site-directed mutagenesis of predicted modification sites

  • Comparison with related proteins with known modifications

Understanding the post-translational regulation of At2g41170 will provide insights into its function and regulation in various physiological contexts.

What troubleshooting steps should I take if I encounter weak or no signal with At2g41170 antibody?

When facing detection challenges with At2g41170 antibody, follow this systematic troubleshooting approach:

• Sample Preparation Issues:

  • Verify protein extraction efficiency (Coomassie staining)

  • Ensure protein is not degraded (fresh protease inhibitors)

  • Check protein transfer efficiency (Ponceau S staining)

  • Consider expression levels and timing (developmental stage, stress responses)

• Antibody-Related Factors:

  • Increase antibody concentration (try 1:500 dilution)

  • Extend incubation time (overnight at 4°C)

  • Verify antibody storage conditions (avoid repeated freeze-thaw)

  • Consider alternative lot or source of antibody

• Detection System Optimization:

  • Use fresh ECL reagents

  • Increase exposure time

  • Try super-sensitive detection reagents

  • Check secondary antibody functionality with other primary antibodies

• Protocol Modifications:

  • Adjust blocking conditions (type, concentration, time)

  • Modify washing steps (duration, buffer composition)

  • Try alternative membranes (PVDF vs. nitrocellulose)

  • Consider native vs. denaturing conditions

Keep detailed records of all troubleshooting steps and results to establish optimal conditions for your specific experimental system.

How can At2g41170 antibody be used in combination with other techniques for comprehensive protein analysis?

Integrating multiple techniques with antibody-based detection provides a more complete understanding of At2g41170:

• Combined Approaches for Functional Analysis:

  • Antibody-based detection + transcriptomic analysis

  • Immunolocalization + fluorescent protein tagging

  • Co-immunoprecipitation + yeast two-hybrid screening

  • Western blotting + mass spectrometry quantification

• Multi-level Experimental Design:

  • Use antibodies to validate findings from genomic screens

  • Combine with CRISPR-Cas9 edited lines for genetic validation

  • Pair with promoter-reporter constructs for expression studies

  • Integrate with metabolomic analysis for downstream effects

• Advanced Imaging Applications:

  • Immunogold electron microscopy for ultrastructural localization

  • Super-resolution microscopy with fluorescent secondary antibodies

  • FRET analysis when combined with fluorescent protein fusions

  • Live cell imaging using cell-permeable tagged antibody fragments

This integrated approach will provide more robust evidence for At2g41170 function and regulation than any single technique alone.

How might At2g41170 relate to plant defense pathways based on current knowledge?

While specific information about At2g41170's role in plant defense is limited in the search results, we can draw some inferences based on related research:

• Possible Defense Connections:

  • The existence of a decoy construct (PK7HFN-AT2G41170decoy) suggests potential involvement in defense mechanisms, as decoy strategies are often employed in studies of plant immunity

  • The search results mention BIK1 (Botrytis-induced kinase 1) , a key defense signaling protein in Arabidopsis, which might provide contextual information about signaling networks At2g41170 could be involved in

• Experimental Approaches to Investigate Defense Roles:

  • Treatment with elicitors (like flg22, which was mentioned for BIK1 studies)

  • Pathogen infection assays with model pathogens

  • Analysis of At2g41170 expression and modification state after pathogen challenge

  • Phenotypic analysis of knockout/overexpression lines during infection

• Potential Relationships to Known Defense Mechanisms:

  • Examine co-expression patterns with established defense genes

  • Assess protein-protein interactions with defense signaling components

  • Investigate subcellular localization changes during immune responses

These approaches would help establish whether At2g41170 plays a direct or indirect role in plant defense pathways.

What considerations should be made when using At2g41170 antibody in immunohistochemistry or immunofluorescence?

While the search results don't specifically mention immunohistochemistry (IHC) or immunofluorescence (IF) applications for At2g41170 antibody, researchers can apply these general considerations:

• Fixation and Tissue Processing:

  • Test multiple fixatives (paraformaldehyde, glutaraldehyde, combination)

  • Optimize fixation time and temperature for plant tissues

  • Consider using fresh frozen sections vs. embedded material

  • Evaluate antigen retrieval methods if necessary

• Antibody Validation for Localization Studies:

  • Use genetically modified plants (knockout/overexpression) as controls

  • Compare patterns with GFP-fusion or other tagged versions

  • Determine optimal antibody concentration (typically higher than for WB)

  • Include all appropriate controls (no primary, pre-immune serum)

• Plant-Specific Considerations:

  • Account for autofluorescence from chlorophyll and cell walls

  • Consider clearing techniques for better visualization

  • Use appropriate counterstains compatible with plant tissues

  • Optimize penetration of antibodies into plant tissues

• Data Interpretation:

  • Collect images from multiple biological replicates

  • Use confocal microscopy for precise subcellular localization

  • Quantify signal where appropriate for comparative studies

  • Consider co-localization with organelle markers

These approaches will help establish reliable localization data for At2g41170 in plant tissues.