GDU6 Antibody

What is GDU6 and what role does it play in Arabidopsis thaliana?

GDU6 (Glutamine Dumper 6) is a protein encoded by the AT3G30725 gene in Arabidopsis thaliana, part of the glutamine dumper family involved in amino acid transport regulation . These proteins play important roles in nitrogen metabolism and allocation within plant tissues. GDU6 is part of a broader network that regulates nutrient transport across cellular membranes, which is essential for plant development and stress responses.

What applications is GDU6 antibody validated for in plant research?

GDU6 antibody (such as product code CSB-PA135647XA01DOA) has been validated for several key applications in plant research:

The antibody is specifically designed to detect GDU6 protein in Arabidopsis thaliana samples, making it valuable for investigating protein expression patterns and functional studies .

What is the typical formulation and storage recommendation for GDU6 antibody?

GDU6 antibody is typically supplied in a liquid formulation containing:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 as a preservative

For optimal stability and performance, the antibody should be:

• Stored at -20°C upon delivery

• Aliquoted to avoid repeated freeze-thaw cycles

• Protected from prolonged exposure to light

• Centrifuged briefly before use to collect contents at the bottom of the tube

How can I validate the specificity of GDU6 antibody in my experiments?

Validating antibody specificity is crucial for experimental reliability. For GDU6 antibody, consider these approaches:

• Positive and negative control samples:

  • Positive control: Wild-type Arabidopsis tissue expressing GDU6

  • Negative control: GDU6 knockout or knockdown lines

• Western blot validation:

  • Confirm a single band at the expected molecular weight

  • Compare band presence/absence in tissues known to express/not express GDU6

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Loss of signal in treated samples confirms specificity

• Orthogonal validation:

  • Compare protein detection results with mRNA expression data

  • If possible, use multiple antibodies targeting different epitopes of GDU6

Importantly, validation should be performed under the same experimental conditions in which the antibody will be used for your research .

What controls should be included when using GDU6 antibody for experimental validation?

When using GDU6 antibody, the following controls should be included to ensure experimental validity:

These controls help distinguish between true GDU6 signals and experimental artifacts, increasing confidence in your results .

How does GDU6 antibody performance compare across different applications?

Based on available research protocols, GDU6 antibody performance varies across applications:

For optimal results in each application, consider the specific sample preparation requirements and detection systems that best complement the antibody characteristics .

What is the optimal protocol for Western blotting with GDU6 antibody in Arabidopsis samples?

For reliable Western blot results with GDU6 antibody in Arabidopsis samples:

• Sample preparation:

  • Homogenize plant tissue in extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitor cocktail)

  • Centrifuge at 12,000g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane (0.22μm) using standard protocols

• Immunodetection:

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

  • Incubate with GDU6 antibody at 1:500-1:1000 dilution in TBST with 1% BSA overnight at 4°C

  • Wash 3x with TBST, 10 minutes each

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

  • Wash 3x with TBST, 10 minutes each

  • Develop using chemiluminescent substrate and image

• Expected results:

  • GDU6 should appear at its predicted molecular weight

  • Validate with appropriate controls as described in section 2.2

How should sample preparation be optimized for GDU6 detection in plant tissues?

Optimal sample preparation is critical for successful GDU6 detection:

• Tissue collection and handling:

  • Harvest fresh tissue and immediately flash-freeze in liquid nitrogen

  • Store at -80°C until processing

  • Grind thoroughly to fine powder while maintaining frozen state

• Extraction buffer optimization:

  • For membrane proteins like GDU6, use buffers containing:

    • Detergents (0.5-1% Triton X-100 or NP-40)

    • Protease inhibitors (complete cocktail)

    • Phosphatase inhibitors (if studying phosphorylation states)

    • Reducing agents (DTT or β-mercaptoethanol)

• Subcellular fractionation:

  • Consider membrane fractionation methods for enrichment

  • Ultracentrifugation can separate membrane-associated proteins

  • Detergent solubilization helps extract membrane proteins

• Protein quantification:

  • Use detergent-compatible protein assay methods

  • Ensure equal loading for comparative analyses

• Sample storage:

  • Add Laemmli buffer with reducing agent immediately after extraction

  • Aliquot to avoid repeated freeze-thaw cycles

  • Store at -20°C for short term or -80°C for long term

What approaches can be used to troubleshoot non-specific binding or weak signal when using GDU6 antibody?

When troubleshooting GDU6 antibody performance issues:

For specific applications like immunoprecipitation, more specialized troubleshooting may be required based on the exact protocol used .

How can I design experiments to study GDU6 protein-protein interactions using the antibody?

To investigate GDU6 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in non-denaturing buffer to preserve protein-protein interactions

  • Incubate lysate with GDU6 antibody coupled to protein A/G beads

  • Wash thoroughly to remove non-specific interactions

  • Elute bound proteins and analyze by Western blot with antibodies against suspected interaction partners

• Proximity ligation assay (PLA):

  • Fix and permeabilize plant tissues

  • Incubate with GDU6 antibody and antibody against candidate interacting protein

  • Apply secondary antibodies with conjugated oligonucleotides

  • If proteins are in close proximity, oligonucleotides will hybridize and can be detected via rolling circle amplification

• Pull-down assays:

  • Follow similar approach to the GDF6 prodomain-CD99 interaction study in the literature:

    • Express recombinant GDU6 with affinity tag

    • Incubate with plant extracts

    • Analyze bound proteins by mass spectrometry

  • Validate interactions using GDU6 antibody in reciprocal experiments

The successful approach used for studying GDF6-CD99 interactions, where microscale thermophoresis revealed a KD of 17.2 nM, provides a good methodological template that could be adapted for GDU6 studies .

What are the key considerations for quantitative analysis of GDU6 expression using antibody-based methods?

For reliable quantitative analysis of GDU6 expression:

• Western blot quantification:

  • Use loading controls appropriate for plant tissues (e.g., actin, tubulin)

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

  • Use technical and biological replicates (minimum n=3)

  • Employ appropriate image analysis software for densitometry

  • Report relative expression normalized to controls

• ELISA-based quantification:

  • Develop a standard curve using recombinant GDU6 protein

  • Validate assay parameters (limits of detection, linear range, precision)

  • Optimize sample dilution to fall within the linear range

  • Include appropriate negative controls

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Consider biological variation in plant samples

  • Report both statistical significance and effect size

• Data presentation:

  • Include representative images alongside quantification

  • Present data with appropriate error bars

  • Clearly state normalization methods and statistical approaches used

How can GDU6 antibody be used to investigate subcellular localization in Arabidopsis tissues?

To investigate GDU6 subcellular localization:

• Immunofluorescence microscopy:

  • Fix plant tissues with 4% paraformaldehyde

  • Permeabilize cell walls and membranes (enzymatic digestion for tougher tissues)

  • Block with 3-5% BSA or normal serum

  • Apply GDU6 antibody at optimized dilution (start with 1:100-1:500)

  • Detect with fluorophore-conjugated secondary antibody

  • Counterstain with subcellular markers (e.g., DAPI for nucleus)

  • Visualize using confocal microscopy

• Subcellular fractionation with Western blot:

  • Prepare subcellular fractions (membrane, cytosolic, nuclear)

  • Verify fraction purity using marker proteins

  • Perform Western blot with GDU6 antibody on each fraction

  • Quantify relative distribution across fractions

• Immuno-electron microscopy:

  • For highest resolution localization studies

  • Prepare samples with appropriate fixation for ultrastructure preservation

  • Apply GDU6 antibody followed by gold-conjugated secondary antibody

  • Visualize using transmission electron microscopy

  • Quantify gold particle distribution relative to cellular structures

This approach parallels methods used for studying other membrane proteins, such as the CD99 internalization that was observed following ligand binding in the GDF6-CD99 system .

How can GDU6 antibody be used to study the effects of environmental stressors on protein expression?

To investigate environmental stress responses:

• Experimental design:

  • Expose plants to controlled stress conditions (drought, salt, cold, heat, pathogens)

  • Collect tissues at multiple time points during stress application and recovery

  • Include appropriate controls (non-stressed plants at each time point)

• Protein expression analysis:

  • Extract proteins using optimized protocols for stressed tissues

  • Quantify GDU6 expression by Western blot or ELISA

  • Normalize to appropriate housekeeping proteins that remain stable under stress conditions

  • Compare expression changes across stress types and intensities

• Correlation with physiological parameters:

  • Measure relevant physiological responses (e.g., water status, ion content)

  • Correlate GDU6 expression with physiological changes

  • Assess statistical relationships between protein levels and stress tolerance

• Integration with other molecular data:

  • Combine protein expression data with transcriptomic analysis

  • Investigate post-translational modifications under stress

  • Develop models of GDU6 function in stress response pathways

What are the methodological considerations for using GDU6 antibody in mutant or transgenic Arabidopsis lines?

When working with mutant or transgenic lines:

• Antibody validation in modified genetic backgrounds:

  • Verify antibody specificity in each genetic background

  • Confirm absence of signal in GDU6 knockout lines

  • For overexpression lines, confirm increased signal intensity

• Comparative expression analysis:

  • Use standardized protein extraction and quantification methods across genotypes

  • Include wild-type controls from similar growth conditions and developmental stages

  • Control for position effects in transgenic lines by analyzing multiple independent lines

• Functional complementation studies:

  • In GDU6 mutant backgrounds, test if phenotypes are rescued by introducing GDU6 variants

  • Use the antibody to confirm expression of the complementing protein

  • Correlate protein expression levels with degree of phenotypic rescue

• Protein interaction changes in mutant backgrounds:

  • Investigate how mutations affect GDU6 interactions with other proteins

  • Compare co-immunoprecipitation results between wild-type and mutant backgrounds

  • Assess changes in subcellular localization in different genetic contexts

How can immunoprecipitation with GDU6 antibody be optimized for downstream proteomics analysis?

For successful immunoprecipitation-mass spectrometry (IP-MS) studies:

• Antibody immobilization and preparation:

  • Covalently link GDU6 antibody to tosyl-activated beads or protein A/G supports

  • Use cross-linking agents to prevent antibody co-elution

  • Include appropriate IgG controls from the same species

• Sample preparation:

  • Extract proteins under non-denaturing conditions

  • Pre-clear lysates with bare beads to reduce non-specific binding

  • Optimize antibody-to-lysate ratios through pilot experiments

• Washing and elution:

  • Use increasingly stringent washing steps to remove non-specific interactions

  • Elute bound proteins using methods compatible with mass spectrometry

  • Consider on-bead digestion for complex samples

• Mass spectrometry considerations:

  • Process samples using LC-MS/MS approaches optimized for plant proteins

  • Use label-free or isotope labeling methods for quantification

  • Search against appropriate Arabidopsis protein databases

  • Apply stringent filtering criteria to identify true interactors

• Data analysis:

  • Compare against negative control IPs to identify specific interactors

  • Consider using probability-based scoring systems for interaction confidence

  • Validate key interactions using alternative methods

This approach has been successfully applied in other systems, such as the study identifying interactions between B1E11K antibody and Plasmodium proteins .

How does the research application of GDU6 antibody compare with other plant membrane protein antibodies?

Comparing GDU6 antibody applications with other plant membrane protein antibodies:

When selecting antibodies for plant membrane protein studies, consider the specific protein family characteristics and available validation data for your experimental system .

What methodological differences exist when using antibodies directed against different domains of membrane proteins?

Working with antibodies against different protein domains requires specific methodological considerations:

• Extracellular domain antibodies:

  • Ideal for flow cytometry and non-permeabilized immunostaining

  • Often useful for blocking functional studies

  • May require different fixation methods to preserve epitopes

  • Example application: Similar to CD99 extracellular domain studies

• Transmembrane domain antibodies:

  • Usually require special detergent extraction methods

  • Often challenging due to hydrophobicity and conserved sequences

  • May need alternative solubilization strategies for Western blot

• Intracellular domain antibodies:

  • Require cell permeabilization for immunostaining

  • Often more specific due to higher sequence variability

  • Better for detecting post-translational modifications

  • Similar to approaches used for studying the CD99 intracellular domain YQKKK motif

• Full-length protein antibodies:

  • May recognize multiple epitopes (polyclonal)

  • Generally more robust to denaturation conditions

  • Could detect degradation products or splice variants

The approach should be tailored based on the specific protein domain targeted by your antibody and the biological question being addressed .

How can researchers integrate antibody-based detection with other molecular techniques to comprehensively study GDU6 function?

For comprehensive functional analysis of GDU6:

• Integrating transcriptomic and proteomic approaches:

  • Compare GDU6 protein expression (antibody-based) with mRNA levels (RT-qPCR/RNA-seq)

  • Identify post-transcriptional regulation mechanisms

  • Correlate with global expression changes during development or stress responses

• Combining with genetic approaches:

  • Use CRISPR/Cas9 or T-DNA insertion lines to generate GDU6 mutants

  • Complement with GDU6 variants and verify expression using the antibody

  • Correlate protein expression with phenotypic outcomes

• Integrating with cell biology techniques:

  • Combine antibody-based localization with fluorescent protein fusions

  • Use super-resolution microscopy for detailed localization studies

  • Correlate protein distribution with functional characteristics

• Metabolomic integration:

  • Link GDU6 expression patterns to changes in amino acid/metabolite profiles

  • Investigate how protein expression correlates with altered nutrient transport

  • Develop models of GDU6's role in nitrogen/amino acid metabolism

• Structural biology connections:

  • Use antibody-validated expression data to inform structural studies

  • Apply structural information to design functional experiments

  • Similar to the approach used in studying GDF6-CD99 interactions, where crystal structure data complemented antibody-based binding studies

This multi-faceted approach provides the most comprehensive understanding of protein function in plant systems.