At5g18200 Antibody

What is the At5g18200 antibody and what protein does it target?

The At5g18200 antibody is a rabbit polyclonal antibody developed against the protein encoded by the AT5G18200 gene in Arabidopsis thaliana. This protein exhibits adenylyltransferase activity, specifically utilizing ADP-glucose as a donor substrate to catalyze the transfer of adenylyl moieties to various phosphate acceptors . The antibody is raised against recombinant Arabidopsis thaliana At5g18200 protein, making it a valuable tool for studying this enzyme in plant research contexts .

What are the basic applications of the At5g18200 antibody?

The At5g18200 antibody has been validated for several standard laboratory applications, including:

• Western Blotting (WB): For detecting the target protein in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of the target protein

• Immunofluorescence (IF): For cellular localization studies (validation may be required)
  These applications provide researchers with versatile options for studying the expression, localization, and function of the At5g18200 protein in plant systems.

What is known about the specificity of the At5g18200 antibody?

The At5g18200 antibody is generated using a specific immunogen consisting of recombinant Arabidopsis thaliana At5g18200 protein. Although primarily reactive with plant species, cross-reactivity testing with other plant species beyond Arabidopsis may be necessary for specific experimental designs. As with all polyclonal antibodies, batch-to-batch variation should be considered, and validation using positive and negative controls is recommended before implementing in critical experiments .

How should I design a Western blot experiment using the At5g18200 antibody?

For optimal Western blot results with the At5g18200 antibody:

• Sample preparation:

  • Extract total protein from plant tissue using a buffer containing protease inhibitors

  • Use approximately 20-50 μg of total protein per lane

  • Include appropriate positive controls (Arabidopsis tissues known to express At5g18200) and negative controls

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation

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

• Blocking and antibody incubation:

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

  • Dilute primary antibody 1:1000 in blocking buffer and incubate overnight at 4°C

  • Wash thoroughly with TBST (at least 3×10 minutes)

  • Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5000 for 1 hour

  • Detect using chemiluminescence substrates appropriate for the expected expression level

What considerations should be made when using the At5g18200 antibody for immunolocalization studies?

For immunolocalization of At5g18200 in plant tissues:

• Tissue fixation and processing:

  • Use 4% paraformaldehyde fixation for 2-4 hours for most plant tissues

  • Consider alternative fixatives such as Farmer's solution for specialized applications

  • Embed in appropriate medium (paraffin for standard histology, or freezing medium for cryosectioning)

• Antigen retrieval:

  • Include an antigen retrieval step (citrate buffer, pH 6.0 at 95°C for 20 minutes) to unmask epitopes

  • Test multiple retrieval methods as plant tissues may require optimization

• Antibody incubation:

  • Dilute antibody 1:200 in blocking buffer (1% BSA in PBS)

  • Incubate sections overnight at 4°C in a humidified chamber

  • Include appropriate controls (primary antibody omission, preimmune serum)

• Detection and imaging:

  • Use fluorescent secondary antibodies for co-localization studies

  • Consider counterstaining with DAPI for nuclear visualization

  • Image using confocal microscopy for subcellular localization

How can I validate the specificity of the At5g18200 antibody for my plant species of interest?

Validating antibody specificity is crucial, especially when working with plant species beyond Arabidopsis:

• Genomic analysis:

  • Perform sequence alignment of At5g18200 protein with homologs from your species of interest

  • Identify conserved regions that might serve as epitopes

• Experimental validation:

  • Western blot analysis comparing wild-type and knockout/knockdown lines (if available)

  • Preabsorption controls using the immunizing peptide

  • Heterologous expression systems to confirm antibody reactivity

• Cross-validation:

  • Compare results with independent methods (RT-qPCR for expression, fluorescent protein fusions for localization)

  • Consider using multiple antibodies targeting different epitopes of the same protein

• Mass spectrometry:

  • Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein

What are common challenges when using plant antibodies like At5g18200 and how can they be addressed?

Plant antibodies often present unique challenges compared to mammalian systems:

How can I minimize non-specific binding when using the At5g18200 antibody in plant tissue immunohistochemistry?

Non-specific binding is a common challenge in plant immunohistochemistry that can be addressed through several strategies:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 2-3 hours at room temperature

  • Consider adding 0.1-0.3% Triton X-100 to improve penetration

• Antibody preparation:

  • Pre-absorb the antibody with plant extract from species lacking the target

  • Purify the antibody using affinity chromatography

  • Use more dilute antibody solutions with longer incubation times

• Washing protocols:

  • Increase washing buffer volume and duration (minimum 5×10 minutes)

  • Include salt (up to 500 mM NaCl) in washing buffers to reduce ionic interactions

  • Add 0.05-0.1% Tween-20 to washing buffers

• Controls and validation:

  • Include absorption controls with immunizing peptide

  • Use knockout/knockdown plant tissues as negative controls

  • Test secondary antibody alone to identify non-specific binding

What are effective methods for preserving and storing the At5g18200 antibody to maintain optimal activity?

Proper storage and handling of antibodies is critical for maintaining their activity:

• Short-term storage (up to 2 weeks):

  • Store at 2-8°C with 0.09% sodium azide as preservative

  • Avoid repeated freeze-thaw cycles

  • Keep in the dark to prevent photobleaching of conjugated fluorophores

• Long-term storage:

  • Aliquot into small volumes to minimize freeze-thaw cycles

  • Store at -20°C for prolonged preservation

  • Add stabilizing proteins like BSA (0.1-1%) if diluted

• Working solutions:

  • Prepare fresh working dilutions on the day of experiment

  • Keep on ice when in use

  • Avoid contamination by using clean pipette tips

• Quality control:

  • Test activity periodically with positive control samples

  • Monitor for signs of degradation (loss of specificity, increased background)

  • Document performance to track potential deterioration over time

How can I use the At5g18200 antibody in combination with other approaches to study protein-protein interactions?

Investigating protein-protein interactions involving At5g18200 requires integrating antibody-based approaches with complementary methods:

• Co-immunoprecipitation (Co-IP):

  • Use the At5g18200 antibody to pull down the target protein complex

  • Analyze precipitated proteins by mass spectrometry or immunoblotting

  • Include appropriate controls (IgG, pre-immune serum)

  • Consider crosslinking to stabilize transient interactions

• Proximity ligation assay (PLA):

  • Combine At5g18200 antibody with antibodies against potential interacting partners

  • Use species-specific PLA probes for detection

  • Optimize fixation and permeabilization for plant tissues

  • Quantify interaction signals using appropriate imaging software

• Chromatin immunoprecipitation (ChIP):

  • Use if At5g18200 may interact with DNA or chromatin-associated proteins

  • Optimize crosslinking conditions for plant tissues

  • Include appropriate controls to validate specificity

  • Analyze precipitated DNA by sequencing or qPCR

• Bimolecular fluorescence complementation (BiFC):

  • Complement antibody studies with protein fragment complementation approaches

  • Express fusion proteins in appropriate plant expression systems

  • Compare results with antibody-based co-localization studies

What are the considerations for using At5g18200 antibody in multiplex immunofluorescence studies?

Multiplex immunofluorescence allows simultaneous detection of multiple proteins, providing valuable insights into protein co-localization and interactions:

• Antibody compatibility:

  • Select antibodies raised in different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider sequential staining with direct labeling

  • Test each antibody individually before combining

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider using quantum dots for narrow emission spectra

  • Account for plant autofluorescence (particularly chlorophyll) when selecting fluorophores

• Protocol optimization:

  • Adjust antibody concentrations individually for balanced signal intensity

  • Optimize antigen retrieval conditions that work for all targeted epitopes

  • Include appropriate absorption controls for each primary antibody

• Image acquisition and analysis:

  • Use sequential scanning to minimize bleed-through

  • Employ spectral unmixing algorithms if necessary

  • Quantify co-localization using appropriate statistical methods (Pearson's coefficient, Manders' overlap)

How can the At5g18200 antibody be utilized in studying protein dynamics during plant development or stress responses?

Analyzing protein dynamics requires strategic experimental design:

• Developmental studies:

  • Sample tissues at defined developmental stages

  • Compare protein levels by quantitative immunoblotting

  • Use immunohistochemistry to track spatial expression patterns

  • Correlate protein levels with transcript abundance via RT-qPCR

• Stress response analysis:

  • Design time-course experiments with appropriate stress treatments

  • Include multiple stress intensities to establish dose-response relationships

  • Monitor protein abundance, modification, and localization changes

  • Compare results across different plant tissues and developmental stages

• Quantitative approaches:

  • Use fluorescent secondary antibodies for quantitative immunofluorescence

  • Employ image analysis software for signal quantification

  • Include internal standards for normalization

  • Present data as relative changes compared to control conditions

• Integration with other methods:

  • Combine with transcriptomics to correlate protein and mRNA levels

  • Use phospho-specific antibodies if phosphorylation may regulate At5g18200

  • Consider pulse-chase experiments to assess protein turnover rates

What are the best practices for quantifying and statistically analyzing At5g18200 protein levels from Western blot data?

Rigorous quantification of Western blot data ensures reliable results:

• Experimental design for quantification:

  • Include a dilution series of positive control samples

  • Load equal amounts of total protein (verify with total protein stains)

  • Run technical replicates across multiple blots

  • Include appropriate housekeeping proteins as loading controls

• Image acquisition:

  • Ensure signals are within the linear dynamic range of detection

  • Avoid saturated pixels that compromise quantification

  • Use the same exposure settings for comparable samples

  • Capture images in standard formats that preserve dynamic range

• Quantification methodology:

  • Use densitometry software (ImageJ, Image Studio, etc.)

  • Subtract local background for each band

  • Normalize to loading controls or total protein stains

  • Calculate relative rather than absolute values when appropriate

• Statistical analysis:

  • Perform experiments with sufficient biological replicates (minimum n=3)

  • Test for normal distribution of data

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Include error bars and p-values in data presentation

How should I interpret unexpected banding patterns when using the At5g18200 antibody in Western blots?

Unexpected bands require systematic investigation:

What approaches can I use to validate that the immunofluorescence signal obtained with the At5g18200 antibody represents the true subcellular localization?

Confirming subcellular localization requires multiple validation strategies:

• Controls and validation:

  • Compare with GFP-fusion protein localization

  • Use genetic knockdown lines as negative controls

  • Perform peptide competition assays

  • Test multiple antibodies against different epitopes of the same protein

• Co-localization with organelle markers:

  • Use established organelle markers in co-staining experiments

  • Calculate co-localization coefficients quantitatively

  • Employ super-resolution microscopy for precise localization

  • Consider 3D reconstruction for complete spatial analysis

• Biochemical fractionation:

  • Perform subcellular fractionation followed by Western blotting

  • Use established markers to confirm fractionation quality

  • Compare results with immunofluorescence data

  • Consider density gradient separation for higher resolution

• Functional validation:

  • Correlate localization with known protein function

  • Test localization under different conditions where function is altered

  • Use site-directed mutagenesis to disrupt targeting sequences

  • Compare wild-type and mutant protein localization

How can the At5g18200 antibody be used to investigate the role of the corresponding protein in ADP-glucose metabolism and plant energy pathways?

Given the adenylyltransferase activity of At5g18200 protein, the antibody can be used to explore its role in energy metabolism:

• Metabolic pathway analysis:

  • Compare protein levels under various energy conditions (light/dark, sugar availability)

  • Correlate protein abundance with metabolite levels (ADP-glucose, downstream products)

  • Investigate co-localization with other enzymes in the pathway

  • Examine protein levels in mutants with altered carbohydrate metabolism

• Enzyme activity correlation:

  • Measure adenylyltransferase activity in tissue extracts

  • Correlate activity levels with protein abundance determined by immunoblotting

  • Investigate post-translational modifications that might regulate activity

  • Compare enzyme kinetics with protein expression patterns

• Protein complex identification:

  • Use the antibody for co-immunoprecipitation to identify interacting partners

  • Determine if the protein functions in a multi-enzyme complex

  • Analyze complex formation under different metabolic conditions

  • Map interaction domains through truncation analysis and immunoprecipitation

What methodological approaches can be used to study potential post-translational modifications of At5g18200 protein using the antibody?

Investigating post-translational modifications (PTMs) requires specialized approaches:

• Electrophoretic mobility analysis:

  • Use Phos-tag or other PTM-sensitive gel systems

  • Compare migration patterns before and after phosphatase treatment

  • Analyze samples from plants treated with PTM-inducing conditions

  • Use 2D electrophoresis to separate modified forms

• Mass spectrometry integration:

  • Immunoprecipitate the protein using the At5g18200 antibody

  • Analyze by LC-MS/MS to identify modification sites

  • Compare modification patterns across different conditions

  • Quantify the stoichiometry of modifications

• PTM-specific antibodies:

  • Generate phospho-specific or other PTM-specific antibodies

  • Use in parallel with the general At5g18200 antibody

  • Perform sequential blotting with general and PTM-specific antibodies

  • Map the cellular distribution of modified forms by immunofluorescence

• Functional validation:

  • Generate site-directed mutants of potential modification sites

  • Express in plant systems and analyze using the At5g18200 antibody

  • Correlate PTM status with protein activity and localization

  • Identify the enzymes responsible for the modifications

How can chromatin immunoprecipitation (ChIP) approaches be adapted for using the At5g18200 antibody to investigate potential DNA interactions?

If At5g18200 has potential chromatin association, ChIP protocols can be adapted:

• ChIP protocol optimization for plant tissues:

  • Optimize crosslinking conditions (formaldehyde concentration and time)

  • Develop tissue-specific nuclear isolation protocols

  • Determine optimal sonication conditions for Arabidopsis chromatin

  • Test antibody concentrations and incubation conditions

• Controls and validation:

  • Include input controls and IgG controls

  • Perform ChIP-qPCR on known targets for validation

  • Compare results from different plant tissues and conditions

  • Use ChIP-seq for genome-wide binding analysis

• Data analysis and interpretation:

  • Identify enriched genomic regions and associated genes

  • Perform motif analysis to identify potential binding sequences

  • Correlate binding sites with gene expression data

  • Compare with ChIP data for known transcription factors or chromatin modifiers

• Functional validation:

  • Perform reporter gene assays for identified target sequences

  • Generate plants with mutations in binding sites using CRISPR/Cas9

  • Analyze expression of target genes in At5g18200 mutant plants

  • Investigate co-localization with other DNA-binding proteins

How might the At5g18200 antibody be utilized in plant biotechnology applications?

The antibody could support various biotechnology applications:

• Metabolic engineering:

  • Monitor At5g18200 protein levels in engineered plants with altered carbon metabolism

  • Use as a marker for successful pathway modification

  • Track protein expression in different plant tissues and developmental stages

  • Correlate protein levels with desired metabolic outputs

• Stress resistance studies:

  • Analyze protein levels under various stress conditions

  • Identify conditions that affect protein abundance or modification

  • Use in screening transgenic plants with altered stress responses

  • Correlate protein function with stress adaptation mechanisms

• Protein production systems:

  • Utilize knowledge of protein structure and function for recombinant expression

  • Optimize extraction and purification protocols based on antibody detection

  • Monitor protein stability in various expression systems

  • Identify optimal conditions for functional protein production

What are potential applications of the At5g18200 antibody in comparative plant biology studies?

The antibody could be valuable for evolutionary and comparative studies:

• Cross-species analysis:

  • Test reactivity with homologous proteins in different plant species

  • Compare protein abundance, localization, and modification across species

  • Identify conserved and divergent aspects of protein function

  • Correlate protein characteristics with ecological adaptations

• Evolutionary studies:

  • Analyze protein conservation in relation to functional constraints

  • Compare post-translational modifications across evolutionary lineages

  • Investigate protein-protein interactions in different species

  • Correlate protein evolution with metabolic pathway evolution

• Adaptation mechanisms:

  • Compare protein expression in plants from different environments

  • Analyze protein changes during acclimation to novel conditions

  • Identify potential selection pressures acting on the protein

  • Correlate protein variants with adaptive phenotypes This FAQ collection provides comprehensive guidance for researchers utilizing the At5g18200 antibody across various experimental contexts, from basic protocols to advanced applications in plant molecular biology research.