At5g15670 Antibody

What is the At5g15670 antibody and what protein does it target?

The At5g15670 antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At5g15670 protein. It specifically recognizes the protein product of the At5g15670 gene, which is identified by Uniprot accession number Q9LFV9 . This antibody is designed for research applications in plant biology, particularly for studies involving Arabidopsis thaliana. Like other research antibodies, it provides a tool for investigating protein expression, localization, and function through various immunological techniques. The antibody is produced through antigen affinity purification methods to ensure specificity, and it is available in liquid form with appropriate storage buffer for maintaining stability .

What are the validated applications for the At5g15670 antibody?

The At5g15670 antibody has been validated for use in enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications . These techniques allow researchers to detect and quantify the target protein in various experimental contexts. For Western blotting, the antibody enables identification of the protein in cell or tissue lysates after separation by gel electrophoresis. ELISA applications permit quantitative analysis of the target protein in solution. While these are the validated applications, researchers may potentially adapt the antibody for other immunological techniques commonly used in plant biology research, such as immunohistochemistry or immunoprecipitation, though additional validation would be required for these applications.

How should the At5g15670 antibody be stored to maintain optimal activity?

For optimal performance and longevity, the At5g15670 antibody should be stored at -20°C or -80°C immediately upon receipt . The antibody is supplied in a storage buffer containing 0.03% Proclin 300 (as preservative), 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during frozen storage . Repeated freeze-thaw cycles should be avoided as they can denature antibody proteins and reduce binding efficacy. When working with the antibody, it is recommended to aliquot the stock solution into smaller volumes prior to storage to minimize the number of freeze-thaw cycles. For short-term use during experimental procedures, the antibody can be kept at 4°C, but should be returned to -20°C or -80°C for long-term storage to prevent degradation.

What controls should be included when designing experiments with the At5g15670 antibody?

Rigorous experimental design for At5g15670 antibody research requires multiple controls to ensure valid and interpretable results. These should include:

• Positive control: Lysate from wild-type Arabidopsis thaliana tissues known to express the At5g15670 protein.

• Negative control: Samples from At5g15670 knockout mutants or tissues where the target protein is not expressed.

• Secondary antibody-only control: Sample processed without primary antibody to assess non-specific binding of the secondary detection system.

• Loading control: Detection of a constitutively expressed protein (e.g., actin or tubulin) to normalize for sample loading variations.

• Pre-absorption control: Primary antibody pre-incubated with excess antigen to demonstrate binding specificity.

This comprehensive control strategy follows established practices in antibody-based research, similar to approaches used with other research antibodies such as those detecting human proteins in clinical studies . The inclusion of these controls helps distinguish between specific signal and background noise, validates antibody specificity, and ensures experimental rigor in plant protein research.

How can optimal working dilutions for Western blot and ELISA be determined?

Determining optimal working dilutions for the At5g15670 antibody requires systematic titration experiments to balance signal strength with background noise. For Western blot applications:

• Prepare a concentration gradient (typically starting from 1:500 to 1:5000) of the primary antibody.

• Run identical protein samples on multiple Western blots.

• Process each blot with a different antibody dilution while keeping all other variables constant.

• Evaluate signal-to-noise ratio for each dilution.

• Select the dilution that provides clear specific bands with minimal background.

For ELISA applications:

• Prepare a standard curve using purified recombinant At5g15670 protein.

• Test multiple antibody dilutions (typically 1:1000 to 1:10,000).

• Calculate the detection limit and linear range for each dilution.

• Select the dilution that provides the optimal balance between sensitivity and specificity.

This methodical approach to antibody dilution optimization is standard practice in immunological research and has been successfully applied with various antibodies across different research contexts . The optimal dilution may vary depending on specific experimental conditions, sample preparation methods, and detection systems used.

What sample preparation methods are recommended for detecting At5g15670 in plant tissues?

Effective detection of At5g15670 in plant tissues requires careful sample preparation to preserve protein integrity while maximizing extraction efficiency. A recommended protocol includes:

• Tissue collection: Harvest fresh Arabidopsis tissues and flash-freeze in liquid nitrogen to prevent protein degradation.

• Homogenization: Grind frozen tissue to a fine powder using a mortar and pestle kept cold with liquid nitrogen.

• Extraction buffer: Extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Extraction procedure:

  • Add 3-5 ml of extraction buffer per gram of tissue powder

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

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

  • Collect supernatant containing soluble proteins

• Protein quantification: Determine protein concentration using Bradford or BCA assay.

• Sample preparation for SDS-PAGE: Mix protein extract with Laemmli buffer containing a reducing agent and heat at 95°C for 5 minutes.

This methodology is adapted from established protocols for plant protein extraction and is designed to maximize the recovery of target proteins while minimizing degradation, similar to approaches used in other plant antibody research contexts . For membrane-associated proteins, modifications to the extraction buffer may be necessary to improve solubilization.

How can the At5g15670 antibody be used to study protein-protein interactions in Arabidopsis?

The At5g15670 antibody can be employed in several advanced techniques to investigate protein-protein interactions:

Co-immunoprecipitation (Co-IP):

• Crosslink proteins in intact plant tissues using formaldehyde (1% for 10 minutes).

• Extract proteins using a non-denaturing buffer to preserve native protein complexes.

• Pre-clear lysate with Protein A/G beads to reduce non-specific binding.

• Incubate lysate with At5g15670 antibody (5-10 μg per mg of total protein).

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

• Wash extensively to remove non-specifically bound proteins.

• Elute and analyze interacting proteins by mass spectrometry.

Proximity-dependent biotin identification (BioID):

• Generate fusion constructs of At5g15670 with a biotin ligase (BirA*).

• Transform Arabidopsis plants with the fusion construct.

• Induce biotinylation of proximal proteins with exogenous biotin.

• Extract proteins and purify biotinylated proteins using streptavidin beads.

• Confirm presence of At5g15670 in complexes using the antibody.

• Identify interaction partners by mass spectrometry.

These methodologies parallel approaches used in other protein interaction studies, such as those investigating antibody-antigen binding in human research contexts , but are adapted specifically for plant systems. The At5g15670 antibody serves as a validation tool to confirm the presence of the target protein in isolated complexes, thereby lending confidence to identified interactions.

What considerations are important when using the At5g15670 antibody for immunolocalization studies?

Successful immunolocalization of At5g15670 requires careful attention to tissue fixation, antigen retrieval, and antibody penetration. A comprehensive approach includes:

Tissue preparation:

• Fix fresh plant tissues in 4% paraformaldehyde in PBS (pH 7.4) for 4-6 hours at 4°C.

• Dehydrate tissues through an ethanol series (30%, 50%, 70%, 90%, 100%).

• Embed in paraffin or suitable resin for sectioning.

• Cut sections at 5-10 μm thickness and mount on adhesive slides.

Antigen retrieval:

• Deparaffinize sections in xylene and rehydrate through ethanol series.

• Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95°C for 20 minutes.

• Cool gradually to room temperature.

Immunostaining protocol:

• Block non-specific binding sites with 5% normal serum in PBS with 0.3% Triton X-100 for 1 hour.

• Incubate with At5g15670 antibody (1:100 to 1:500 dilution) overnight at 4°C.

• Wash three times with PBS containing 0.1% Tween-20.

• Incubate with fluorophore-conjugated secondary antibody for 1-2 hours at room temperature.

• Counterstain nuclei with DAPI (1 μg/ml) for 10 minutes.

• Mount in anti-fade medium and image using confocal microscopy.

Critical controls:

• Omission of primary antibody

• Pre-absorption with recombinant antigen

• Tissues from At5g15670 knockout plants

This methodology draws on established immunohistochemical approaches used in various research contexts, including those studying epitope recognition in human antibody research . The protocol is specifically adapted for plant tissues, which present unique challenges such as cell wall barriers and autofluorescence that must be addressed for successful immunolocalization.

How can quantitative analysis of At5g15670 expression be performed across different developmental stages?

Quantitative analysis of At5g15670 expression across developmental stages requires integration of multiple techniques:

Quantitative Western Blot Analysis:

• Collect tissue samples from different developmental stages (e.g., seedling, juvenile, mature, flowering, senescent).

• Extract total protein using a standardized protocol.

• Load equal amounts of protein (20-30 μg) per lane.

• Include a dilution series of recombinant At5g15670 protein (0.1-10 ng) as a standard curve.

• Probe with At5g15670 antibody at optimized dilution.

• Normalize signal to internal loading controls (e.g., ACTIN-7) .

• Quantify band intensity using densitometry software.

ELISA-based Quantification:

• Coat microplate wells with capture antibody against At5g15670.

• Add protein extracts from different developmental stages.

• Detect bound protein using a detection antibody system.

• Generate standard curve using purified recombinant protein.

• Calculate absolute protein concentration in each sample.

Data Integration and Visualization:

This approach to quantitative protein analysis across developmental stages is comparable to methodologies employed in other antibody-based research studies , adapted specifically for plant developmental biology research with the At5g15670 antibody.

What are common issues when using the At5g15670 antibody and how can they be resolved?

Researchers working with the At5g15670 antibody may encounter several technical challenges. The following table outlines common issues and proposed solutions:

This troubleshooting approach parallels methods used in other antibody research contexts, such as those investigating human antibodies , but is specifically tailored to plant protein research with the At5g15670 antibody. Systematic troubleshooting is essential for generating reliable and reproducible data in plant immunological research.

How can potential cross-reactivity of the At5g15670 antibody be assessed and addressed?

Evaluating and addressing potential cross-reactivity of the At5g15670 antibody requires a multi-faceted approach:

Sequence-based assessment:

• Perform BLAST analysis of the immunogen sequence against the Arabidopsis proteome to identify proteins with sequence similarity.

• Predict potential cross-reactive epitopes using epitope prediction algorithms.

• Compare predicted epitopes with proteins expressed in tissues of interest.

Experimental validation:

• Western blot analysis with multiple tissues:

  • Compare band patterns across different tissues with varying protein expression profiles.

  • Look for unexpected bands that don't match the predicted molecular weight.

• Competitive binding assays:

  • Pre-incubate antibody with excess recombinant At5g15670 protein.

  • Compare results with non-absorbed antibody to identify non-specific binding.

• Knockout validation:

  • Test antibody against tissues from verified At5g15670 knockout plants.

  • Any remaining signal indicates potential cross-reactivity.

• Mass spectrometry validation:

  • Perform immunoprecipitation with the At5g15670 antibody.

  • Analyze precipitated proteins by mass spectrometry.

  • Identify any co-precipitated proteins that may represent cross-reactivity.

This methodical approach to cross-reactivity assessment is consistent with best practices in antibody validation across research fields and has been adapted specifically for plant antibody research applications.

How should data conflicts between At5g15670 antibody results and other experimental methods be reconciled?

When faced with conflicting data between At5g15670 antibody results and other experimental methods, researchers should employ a systematic reconciliation approach:

• Identify the nature of the conflict:

  • Presence/absence discrepancy (e.g., antibody detects protein but RNA-seq shows no transcript)

  • Quantitative discrepancy (e.g., antibody shows high protein levels but qPCR indicates low mRNA)

  • Localization discrepancy (e.g., antibody shows nuclear localization but GFP fusion shows cytoplasmic)

• Consider biological explanations:

  • Post-transcriptional regulation might explain discrepancies between mRNA and protein levels

  • Post-translational modifications could affect antibody recognition

  • Protein stability and turnover may not correlate with transcript levels

  • Protein trafficking or processing might explain localization differences

• Technical validation:

  • Repeat experiments with additional controls

  • Use alternative antibody lots or sources if available

  • Validate findings with orthogonal methods (e.g., mass spectrometry)

  • Perform epitope mapping to understand antibody binding characteristics

• Integrated analysis framework:

This framework for reconciling conflicting data is based on established approaches to resolving experimental discrepancies in biological research , adapted specifically for research involving the At5g15670 antibody in plant systems.

How can the At5g15670 antibody be used in chromatin immunoprecipitation (ChIP) studies?

While not explicitly validated for ChIP applications, the At5g15670 antibody may be adapted for chromatin immunoprecipitation studies with appropriate optimization. A recommended protocol includes:

• Crosslinking and chromatin preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10 minutes under vacuum.

  • Quench with 0.125 M glycine for 5 minutes.

  • Extract nuclei using a nuclear isolation buffer.

  • Sonicate chromatin to generate fragments of 200-500 bp.

• Antibody optimization:

  • Test different amounts of At5g15670 antibody (2-10 μg per ChIP reaction).

  • Include IgG control antibody at equivalent concentrations.

  • Validate specificity using tissue from At5g15670 knockout plants.

• Immunoprecipitation:

  • Pre-clear chromatin with Protein A/G beads.

  • Incubate cleared chromatin with At5g15670 antibody overnight at 4°C.

  • Capture antibody-chromatin complexes with Protein A/G beads.

  • Wash extensively to remove non-specific binding.

• DNA recovery and analysis:

  • Reverse crosslinks at 65°C overnight.

  • Treat with RNase A and Proteinase K.

  • Purify DNA using phenol-chloroform extraction or commercial kits.

  • Analyze enrichment by qPCR or next-generation sequencing.

This methodological approach draws on ChIP protocols adapted for plant systems and antibody-based chromatin research in other contexts , modified specifically for potential applications of the At5g15670 antibody in plant epigenetics research.

What considerations are important when using the At5g15670 antibody in plant stress response studies?

The At5g15670 antibody can be a valuable tool for investigating protein dynamics during plant stress responses, but several important considerations must be addressed:

• Baseline expression profiling:

  • Establish normal expression patterns across tissues and developmental stages.

  • Create a reference dataset of protein levels under standard growth conditions.

  • Determine natural variation in expression to distinguish from stress-induced changes.

• Stress treatment standardization:

  • Define precise stress application protocols (duration, intensity, conditions).

  • Include recovery phases to assess reversibility of protein changes.

  • Apply multiple stress types to test specificity of responses.

• Time-course analysis:

  • Design sampling intervals appropriate to the stress type (minutes to days).

  • Process all samples simultaneously to minimize technical variation.

  • Include early time points to capture immediate responses.

• Subcellular fractionation:

  • Monitor potential changes in protein localization during stress.

  • Employ differential centrifugation to isolate cellular compartments.

  • Validate fraction purity with compartment-specific markers.

• Post-translational modification analysis:

  • Assess potential stress-induced modifications (phosphorylation, ubiquitination).

  • Use phosphatase treatments to identify mobility shifts due to phosphorylation.

  • Compare reduced and non-reduced samples to identify disulfide bond formation.

This systematic approach to stress response studies using the At5g15670 antibody follows established methodologies in plant stress biology research, incorporating best practices from antibody-based protein studies in various research contexts .

How can the At5g15670 antibody be integrated with emerging technologies for comprehensive protein analysis?

Integration of the At5g15670 antibody with cutting-edge technologies enables more comprehensive protein analysis:

Single-cell proteomics applications:

• Optimize antibody for compatibility with cell fixation and permeabilization protocols.

• Validate signal specificity at single-cell resolution using confocal microscopy.

• Combine with cell-type specific markers for co-localization studies.

• Integrate with flow cytometry for quantitative single-cell protein measurements.

Proximity labeling approaches:

• Use the antibody to validate proximity labeling results from BioID or APEX2 fusion experiments.

• Confirm identification of interaction partners through co-immunoprecipitation.

• Correlate antibody-detected protein levels with proximity labeling efficiency.

Multiplexed imaging technologies:

• Combine At5g15670 antibody with other validated antibodies for simultaneous detection.

• Optimize antibody labeling with appropriate fluorophores or metal tags for multiplexed imaging.

• Develop protocols compatible with Imaging Mass Cytometry or CODEX multiplexed imaging.

Integration with -omics platforms:

• Correlate antibody-detected protein levels with:

  • Transcriptomics data from RNA-seq

  • Proteomics data from mass spectrometry

  • Metabolomics profiles related to protein function

• Develop computational frameworks to integrate multi-omics data with antibody-based measurements.

This forward-looking approach to integrating the At5g15670 antibody with emerging technologies parallels advanced research methods being developed in other fields , adapted specifically for plant biology research applications.