At4g25220 Antibody

What is the At4g25220 protein and its function in Arabidopsis thaliana?

The At4g25220 gene encodes a glycerol-3-phosphate permease (G3Pp2), which is part of the phosphate starvation-induced glycerol-3-phosphate permease gene family in Arabidopsis thaliana . This protein functions as a transporter involved in transmembrane transport processes and plays a significant role in phosphate ion homeostasis . It belongs to the Major Facilitator Superfamily (MFS) of transporters and is specifically expressed in root hairs and roots, suggesting a specialized function in these tissues . The gene is located on chromosome 4 at position 12921161-12922762 on the positive strand .

What are the key specifications of commercially available At4g25220 Antibodies?

At4g25220 Antibodies are typically polyclonal antibodies raised in rabbits using recombinant Arabidopsis thaliana At4g25220 protein as the immunogen . These antibodies are usually supplied in liquid form with a storage buffer containing preservatives (such as 0.03% Proclin 300) and stabilizers (like 50% Glycerol in 0.01M PBS, pH 7.4) . They are purified using antigen affinity methods and are intended for research applications such as ELISA and Western Blot . The antibodies specifically target the At4g25220 protein (UniProt: Q9SB41) from Arabidopsis thaliana and are designed for research use only, not for diagnostic or therapeutic procedures .

How should At4g25220 Antibody be stored and handled for optimal performance?

For optimal performance and stability, At4g25220 Antibody should be stored at -20°C or -80°C upon receipt . Researchers should avoid repeated freeze-thaw cycles as this can degrade antibody quality and affect binding efficiency. When working with the antibody, maintain cold chain protocols by keeping it on ice or in refrigerated conditions during experiments. The antibody is typically supplied in a stabilizing buffer containing glycerol (50%) and PBS (0.01M, pH 7.4) with preservatives like Proclin 300 (0.03%) , which helps maintain its structural integrity during storage and handling.

What are the validated applications for At4g25220 Antibody in plant research?

The At4g25220 Antibody has been validated for several experimental applications in plant research. These include Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) for the detection and quantification of the target protein . When designing experiments using this antibody, researchers should consider the specific experimental conditions that have been validated. For Western Blotting, optimal dilutions typically range from 1:500 to 1:2000, depending on the detection system and sample concentration. For ELISA, researchers should follow standardized protocols with appropriate controls to ensure accurate identification of the antigen .

How can I verify the specificity of At4g25220 Antibody in my experiments?

Verifying antibody specificity is crucial for reliable experimental results. For At4g25220 Antibody, researchers should implement multiple verification strategies:

• Positive and Negative Controls: Include known positive samples (e.g., Arabidopsis thaliana wild-type extracts) and negative controls (e.g., knockout or knockdown lines for AT4G25220 gene) .

• Peptide Competition Assay: Pre-incubate the antibody with its specific immunogenic peptide before application to your sample. Significant reduction in signal indicates specificity for the target epitope .

• Mass Spectrometry Validation: Perform immunoprecipitation followed by mass spectrometric analysis to confirm that the antibody is pulling down the correct protein .

• Multiple Antibody Approach: Use different antibodies targeting different epitopes of the same protein to confirm results .

• Cross-Reactivity Testing: Test the antibody against related plant species to assess potential cross-reactivity with homologous proteins .

The importance of these verification steps is underscored by research demonstrating that antibodies can exhibit unexpected cross-reactivity, as seen with certain anti-glucocorticoid receptor antibodies that were found to predominantly bind to unintended proteins like AMPD2 and TRIM28 .

What are the optimal conditions for Western Blot using At4g25220 Antibody?

For optimal Western Blot results with At4g25220 Antibody:

• Sample Preparation: Extract proteins from root tissues where At4g25220 is predominantly expressed . Use a buffer containing protease inhibitors to prevent degradation.

• Gel Electrophoresis: Use 10-12% SDS-PAGE gels for optimal separation of the target protein.

• Transfer Conditions: Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight at 4°C.

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

• Primary Antibody Incubation: Dilute At4g25220 Antibody 1:1000 in blocking solution and incubate overnight at 4°C.

• Washing: Wash 3-5 times with TBST, 5 minutes each.

• Secondary Antibody: Use anti-rabbit IgG conjugated with HRP at a 1:5000 dilution for 1 hour at room temperature.

• Detection: Use enhanced chemiluminescence (ECL) reagents for detection.

• Controls: Include a positive control (Arabidopsis wild-type root extract) and a negative control (AT4G25220 knockout/knockdown line) .

How can I investigate At4g25220 protein interactions using immunoprecipitation?

Investigating protein interactions involving At4g25220 requires careful experimental design:

• Buffer Selection: Use a mild lysis buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors) to preserve protein-protein interactions.

• Cross-linking (Optional): Consider using formaldehyde (1%) or DSP (dithiobis(succinimidyl propionate)) to stabilize transient interactions.

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

• Antibody Incubation: Incubate cleared lysates with At4g25220 Antibody (2-5 μg per 1 mg of total protein) overnight at 4°C with gentle rotation.

• Bead Capture: Add Protein A or Protein G beads for 1-2 hours at 4°C to capture antibody-protein complexes.

• Washing: Perform stringent washing (at least 5 times) with wash buffers of increasing stringency.

• Elution and Analysis: Elute bound proteins for downstream analysis by SDS-PAGE followed by Western blotting or mass spectrometry .

• Controls: Include an IgG control to identify non-specific interactions and perform a reverse immunoprecipitation with antibodies against suspected interacting partners .

• Validation: Confirm interactions using alternative methods such as yeast two-hybrid or bimolecular fluorescence complementation.

What approaches can be used to study At4g25220 localization in plant cells?

To study the subcellular localization of At4g25220:

• Immunofluorescence Microscopy:

  • Fix plant tissues with 4% paraformaldehyde

  • Permeabilize with a detergent like Triton X-100

  • Block with 3% BSA in PBS

  • Incubate with At4g25220 Antibody (1:100-1:500 dilution)

  • Use fluorophore-conjugated secondary antibodies for detection

  • Include organelle markers for co-localization studies

• GFP Fusion Proteins:

  • Generate At4g25220-GFP fusion constructs

  • Express in plant tissues via Agrobacterium-mediated transformation

  • Visualize using confocal microscopy

  • Compare with immunofluorescence results to validate findings

• Subcellular Fractionation:

  • Isolate different cellular compartments (membrane, cytosol, etc.)

  • Analyze fractions by Western blot using At4g25220 Antibody

  • Include controls for each fraction (e.g., membrane markers)

• Electron Microscopy:

  • Use immunogold labeling with At4g25220 Antibody

  • Visualize at ultrastructural level

Based on gene annotation, At4g25220 (G3Pp2) is expected to localize to cellular membranes, particularly in root hair cells, consistent with its function as a transmembrane transporter in the Major Facilitator Superfamily .

How can quantitative proteomics be integrated with At4g25220 Antibody studies?

Integrating quantitative proteomics with At4g25220 Antibody studies can provide comprehensive insights into protein abundance, modifications, and interactions:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Perform IP using At4g25220 Antibody

  • Process samples for LC-MS/MS analysis

  • Identify interacting proteins and post-translational modifications

  • Use label-free quantification or isotopic labeling (SILAC, TMT) for relative quantification

  • Compare results with control IPs to filter out non-specific interactions

• Targeted Proteomics (MRM/PRM):

  • Develop Multiple Reaction Monitoring (MRM) or Parallel Reaction Monitoring (PRM) assays

  • Quantify At4g25220 protein levels across different conditions or tissues

  • Use heavy-labeled peptide standards for absolute quantification

• Proximity-Dependent Biotin Identification (BioID):

  • Create fusion proteins of At4g25220 with a biotin ligase

  • Identify proximal proteins through biotinylation

  • Validate interactions using At4g25220 Antibody

• Phosphoproteomics:

  • Enrich for phosphopeptides after At4g25220 immunoprecipitation

  • Identify phosphorylation sites and their regulation

  • Connect to signaling pathways relevant to phosphate homeostasis

How can I address cross-reactivity issues with At4g25220 Antibody?

Cross-reactivity is a common challenge with antibodies that can lead to misleading results. To address potential cross-reactivity with At4g25220 Antibody:

• Epitope Analysis: Review the immunogenic sequence used to generate the antibody and perform in silico analysis to identify potential cross-reactive proteins with similar epitopes .

• Peptide Blocking: Pre-incubate the antibody with its specific peptide epitope before use. This should significantly reduce binding to the target protein while minimally affecting non-specific binding . Mass spectrometric analysis of immunoprecipitation samples with and without peptide blocking can quantitatively assess specific vs. non-specific binding, as demonstrated in studies with other antibodies .

• Knockout/Knockdown Validation: Test the antibody on samples from At4g25220 knockout or knockdown plants. Persistence of signal indicates cross-reactivity .

• Multiple Antibody Approach: Use alternative antibodies targeting different epitopes of At4g25220 and compare results .

• Mass Spectrometry Validation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins pulled down by the antibody .

The caution regarding cross-reactivity is supported by research showing that even well-established antibodies, like the anti-glucocorticoid receptor antibody clone 5E4, can exhibit unexpected binding to unrelated proteins such as AMPD2 and TRIM28 .

What are the best practices for validating At4g25220 Antibody in different experimental systems?

To ensure reliable results across different experimental systems:

• Initial Validation Panel:

  • Western blot with positive controls (tissues known to express At4g25220)

  • Negative controls (knockout/knockdown lines)

  • Peptide competition assay

  • Testing against related species for cross-reactivity

• Application-Specific Validation:

  • For immunohistochemistry: Include isotype controls

  • For ELISA: Generate standard curves with recombinant protein

  • For IP: Confirm pull-down by Western blot and mass spectrometry

  • For flow cytometry: Use appropriate controls for gating

• Lot-to-Lot Validation:

  • Test new antibody lots against previous lots

  • Maintain reference samples for comparison

  • Document optimal working dilutions for each application

• Species Cross-Reactivity Assessment:

  • Test antibody reactivity on homologous proteins from related plant species

  • Perform sequence alignment of the epitope region across species

• External Validation:

  • Compare results with published literature

  • Consider orthogonal approaches that don't rely on antibodies

These validation steps are particularly important given recent concerns about antibody specificity in the research community, as highlighted by studies exposing unspecific binding of established reagents .

How can I optimize immunoprecipitation protocols for low-abundance At4g25220 protein?

Optimizing immunoprecipitation for low-abundance proteins like At4g25220:

• Sample Enrichment:

  • Start with tissues known to have higher expression (roots and root hairs for At4g25220)

  • Consider using plants grown under phosphate starvation conditions to potentially upregulate expression, as At4g25220 is part of the phosphate starvation-induced gene family

  • Scale up starting material (use more tissue)

• Lysis Optimization:

  • Test different lysis buffers to maximize protein extraction

  • Use mechanical disruption methods optimized for plant tissues

  • Include appropriate protease inhibitors to prevent degradation

• Antibody Amount Optimization:

  • Titrate antibody amounts to find the optimal concentration

  • Consider using higher antibody concentrations (5-10 μg per mg of lysate)

  • Increase incubation time to overnight at 4°C

• Bead Optimization:

  • Compare different types of beads (magnetic vs. agarose)

  • Pre-coat beads with antibody before adding lysate

  • Optimize bead volume and incubation time

• Detection Enhancement:

  • Use highly sensitive ECL substrates for Western blot detection

  • Consider amplification systems for detection

  • Use more sensitive mass spectrometry approaches for protein identification

• Cross-linking:

  • Use DSP or formaldehyde cross-linking to stabilize interactions

  • Optimize cross-linking conditions to maintain specificity

• Elution Strategies:

  • Compare different elution methods (low pH, high salt, SDS, peptide competition)

  • Optimize elution conditions to maximize recovery

• Control Experiments:

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

  • Use samples from knockout/knockdown plants as specificity controls

How should I analyze Western blot data using At4g25220 Antibody for quantitative comparisons?

For quantitative Western blot analysis using At4g25220 Antibody:

• Sample Preparation Consistency:

  • Use equal amounts of total protein for each sample (validate by total protein staining)

  • Prepare all samples identically to minimize technical variation

• Controls and Normalization:

  • Include loading controls appropriate for your experimental system (e.g., actin, tubulin, or GAPDH)

  • Consider using total protein normalization methods (e.g., Stain-Free technology or Ponceau staining)

  • Include a standard curve using recombinant At4g25220 protein if absolute quantification is needed

• Data Acquisition:

  • Use a digital imaging system with a linear dynamic range

  • Avoid saturation of signals

  • Capture multiple exposures to ensure linearity of signal

• Quantification Method:

  • Use densitometry software to quantify band intensity

  • Define consistent measurement areas across all lanes

  • Subtract background signal appropriately

• Statistical Analysis:

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

  • Apply appropriate statistical tests based on your experimental design

  • Report both statistical significance and effect size

• Data Presentation:

  • Present normalized data with error bars

  • Indicate statistical significance clearly

  • Show representative blot images alongside quantification

• Technical Considerations:

  • Be aware of potential cross-reactivity issues that might affect quantification

  • Validate antibody specificity before quantitative comparisons

  • Consider the linear dynamic range of your detection method

What statistical approaches are recommended for analyzing At4g25220 expression across different experimental conditions?

For statistical analysis of At4g25220 expression data:

• Exploratory Data Analysis:

  • Begin with visual inspection of data distribution

  • Check for outliers and normality

  • Consider transformations if data is skewed

• Comparative Analysis Between Two Conditions:

  • Student's t-test for normally distributed data

  • Mann-Whitney U test for non-parametric data

  • Calculate effect sizes (Cohen's d or similar) in addition to p-values

• Multiple Condition Comparisons:

  • ANOVA followed by post-hoc tests (Tukey's HSD, Bonferroni) for normally distributed data

  • Kruskal-Wallis followed by Dunn's test for non-parametric data

  • Control for multiple testing (Benjamini-Hochberg correction)

• Time Course Experiments:

  • Repeated measures ANOVA for parametric data

  • Friedman test for non-parametric data

  • Consider mixed effects models for complex designs

• Correlation Analysis:

  • Pearson correlation for linear relationships

  • Spearman correlation for monotonic non-linear relationships

  • Consider partial correlations to control for confounding variables

• Regression Models:

  • Linear regression for continuous predictors

  • Logistic regression for binary outcomes

  • Multiple regression for complex relationships

• Power Analysis:

  • Determine appropriate sample sizes before experiments

  • Calculate post-hoc power for completed experiments

  • Consider biological significance in addition to statistical significance

• Data Visualization:

  • Use box plots or violin plots to show distribution

  • Include individual data points for transparency

  • Create clear, informative figures with appropriate legends

How can multi-omics approaches be integrated with At4g25220 Antibody studies to elucidate G3Pp2 function?

Integrating multi-omics approaches with At4g25220 Antibody studies can provide comprehensive understanding of G3Pp2 function:

• Proteomics + Transcriptomics:

  • Compare protein levels (using At4g25220 Antibody) with mRNA expression

  • Identify post-transcriptional regulation mechanisms

  • Study translational efficiency under different conditions

• Proteomics + Metabolomics:

  • Correlate G3Pp2 protein levels with glycerol-3-phosphate and phosphate metabolite profiles

  • Identify metabolic pathways influenced by G3Pp2 function

  • Map metabolic changes under phosphate starvation conditions

• Proteomics + Interactomics:

  • Use At4g25220 Antibody for immunoprecipitation followed by mass spectrometry

  • Identify protein-protein interaction networks

  • Map G3Pp2 to specific cellular complexes and pathways

• Proteomics + Phenomics:

  • Correlate G3Pp2 protein levels with phenotypic traits

  • Connect molecular mechanisms to physiological outcomes

  • Identify biomarkers for phosphate stress responses

• Systems Biology Integration:

  • Create predictive models incorporating multi-omics data

  • Identify regulatory networks controlling G3Pp2 expression and function

  • Simulate responses to environmental changes

• Spatial Proteomics:

  • Combine antibody-based imaging with spatial transcriptomics

  • Map G3Pp2 distribution in different cell types and tissues

  • Correlate with tissue-specific metabolic profiles

These integrated approaches can help elucidate how G3Pp2 contributes to phosphate ion homeostasis and responds to environmental stresses in root tissues.

What are potential research applications of At4g25220 Antibody in studying plant stress responses?

At4g25220 Antibody offers valuable applications for studying plant stress responses:

• Phosphate Starvation Studies:

  • Track G3Pp2 protein dynamics during phosphate deprivation

  • Compare with other members of the phosphate starvation-induced glycerol-3-phosphate permease gene family

  • Identify regulatory mechanisms controlling G3Pp2 expression under stress

• Root Architecture Analysis:

  • Examine G3Pp2 protein distribution in different root zones during stress

  • Correlate with root hair development under varying phosphate conditions

  • Study the role of G3Pp2 in root adaptations to nutrient availability

• Membrane Transport Studies:

  • Investigate G3Pp2's role in membrane transport during stress

  • Study how phosphate and glycerol-3-phosphate transport is regulated

  • Examine changes in subcellular localization under stress conditions

• Cross-Talk Between Stress Pathways:

  • Study G3Pp2 response to multiple simultaneous stresses

  • Investigate regulation by different stress-responsive transcription factors

  • Identify signaling pathways converging on G3Pp2 regulation

• Genetic Variation Analysis:

  • Use At4g25220 Antibody to compare G3Pp2 protein levels across ecotypes

  • Correlate with natural variation in stress tolerance

  • Identify post-translational modifications induced by stress

• Transgenic Studies:

  • Compare protein levels in overexpression and knockdown lines

  • Correlate with phenotypic outcomes under stress

  • Validate antibody specificity using genetic tools

These applications leverage At4g25220's role as part of the phosphate starvation-induced glycerol-3-phosphate permease gene family, which suggests its importance in plant responses to nutrient deficiency stresses .