AGD15 Antibody

What is AGD15 Antibody and what cellular proteins does it target?

AGD15 Antibody targets ADP ribosylation factor (ARF) and functions as a GTPase-activating protein (GAP). It specifically recognizes the protein encoded by the At3g17660 gene. The antibody has several synonyms in scientific literature, including At3g17660 antibody, MKP6.22 Probable ADP-ribosylation factor GTPase-activating protein AGD15 antibody, ARF GAP AGD15 antibody, Protein ARF-GAP DOMAIN 15 antibody, and AtAGD15 antibody.
ARF proteins play crucial roles in membrane trafficking and vesicle formation in eukaryotic cells. By targeting these proteins, AGD15 Antibody enables researchers to study regulatory mechanisms involving GTPase activity that are essential for cellular function.

How should researchers prepare and store AGD15 Antibody to maintain optimal activity?

For optimal activity maintenance, AGD15 Antibody should be stored according to these guidelines:

• Long-term storage: -20°C to -70°C for up to 12 months from receipt date

• Medium-term storage: 2°C to 8°C under sterile conditions for up to 1 month after reconstitution

• Extended storage post-reconstitution: -20°C to -70°C for up to 6 months under sterile conditions
  To maintain antibody integrity, researchers should:

• Use a manual defrost freezer

• Avoid repeated freeze-thaw cycles

• Store the antibody in buffer containing preservatives like 0.03% Proclin 300

• Use buffer constituents such as 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
  These storage guidelines parallel general monoclonal antibody handling protocols that help preserve antibody function and specificity for experimental applications.

What are the recommended methodologies for validating AGD15 Antibody specificity prior to experimental use?

Rigorous validation of AGD15 Antibody specificity should follow established protocols for antibody validation:

• Expression systems validation:

  • Test antibody against lysates from systems expressing recombinant AGD15

  • Compare with negative controls lacking the target protein

  • Analyze protein bands at expected molecular weights

• Cross-reactivity assessment:

  • Perform tissue cross-reactivity studies using appropriate species tissues including human samples

  • Test against related proteins with similar structural domains

  • Document any non-specific binding patterns

• Knockout/knockdown validation:

  • Compare signals between wild-type and AGD15-depleted samples

  • Quantify signal-to-noise ratios and establish statistical significance

• Immunoprecipitation analysis:

  • Conduct pull-down assays followed by mass spectrometry

  • Identify and quantify target enrichment versus non-specific binding

• Multiple detection methods:

  • Use complementary techniques (Western blotting, immunofluorescence)

  • Compare localization patterns with predicted protein distribution
    These methodological approaches align with antibody validation standards outlined in preclinical development protocols for therapeutic antibodies .

How can AGD15 Antibody be incorporated into immunofluorescence experiments to study subcellular localization?

For successful immunofluorescence experiments using AGD15 Antibody, researchers should follow this methodological framework:

• Sample preparation:

  • Fix samples using 4% paraformaldehyde or other appropriate fixative

  • Permeabilize with 0.1-0.3% Triton X-100 to enable antibody access to intracellular targets

  • Perform antigen retrieval if necessary to expose epitopes

• Blocking optimization:

  • Use 5-10% normal serum or BSA in PBS to minimize non-specific binding

  • Include 0.1% detergent in blocking solution to reduce background

  • Block for at least 1 hour at room temperature

• Primary antibody incubation:

  • Determine optimal dilution through titration experiments (starting range: 1:100-1:1000)

  • Incubate at 4°C overnight for maximum sensitivity

  • Include appropriate negative controls (no primary antibody, isotype control)

• Detection and visualization:

  • Use fluorescently labeled secondary antibodies specific to the host species of AGD15 Antibody

  • Include nuclear counterstain for contextual reference

  • Employ confocal microscopy for precise subcellular localization

• Colocalization analysis:

  • Perform dual labeling with established organelle markers

  • Calculate Pearson's correlation coefficients for quantitative assessment

  • Generate intensity correlation plots to visualize spatial relationships
    This approach provides robust visualization of AGD15 distribution while minimizing artifacts and non-specific signals.

How can researchers use AGD15 Antibody in conjunction with mass spectrometry to identify protein interaction networks?

Integrating AGD15 Antibody with mass spectrometry for interaction network analysis requires careful methodological planning:

• Immunoprecipitation optimization:

  • Determine optimal buffer conditions that preserve protein-protein interactions

  • Test different lysis methods to maximize recovery while maintaining complex integrity

  • Establish appropriate antibody-to-lysate ratios through preliminary experiments

• Control experiments:

  • Include IgG control immunoprecipitations to identify non-specific binders

  • Perform parallel experiments with samples lacking AGD15 expression

  • Use isotope labeling approaches (SILAC, TMT) for quantitative comparison

• Sample processing:

  • Employ on-bead digestion to minimize sample loss

  • Consider crosslinking approaches to capture transient interactions

  • Implement peptide fractionation to increase proteome coverage

• Mass spectrometry analysis:

  • Use high-resolution instruments for accurate protein identification

  • Implement both data-dependent and data-independent acquisition modes

  • Develop specific search parameters for post-translational modifications

• Data analysis and validation:

  • Apply stringent statistical filters to distinguish true interactors from background

  • Validate key interactions through reciprocal immunoprecipitations

  • Perform functional studies on identified interaction partners
    This integrated approach aligns with contemporary proteomics workflows used in antibody research and development .

What are the critical considerations when interpreting conflicting results between AGD15 protein levels detected by antibody and mRNA expression data?

When faced with discrepancies between AGD15 Antibody detection and mRNA expression, researchers should consider these analytical frameworks:

• Biological regulation assessment:

  • Evaluate potential post-transcriptional regulation mechanisms

  • Consider protein stability and turnover rates

  • Assess the impact of post-translational modifications on epitope recognition

• Technical validation:

  • Verify antibody specificity using multiple controls

  • Confirm mRNA measurements with alternative primers/probes

  • Test different antibody lots and storage conditions

• Experimental design analysis:

  • Review temporal aspects (protein synthesis lag following transcription)

  • Consider spatial differences (subcellular compartmentalization)

  • Examine developmental or condition-specific regulatory mechanisms

• Quantitative comparison:

  Analysis Method	Advantages	Limitations	Resolution Approach
  Western blot	Direct protein detection	Semi-quantitative	Include loading controls and standard curves
  qRT-PCR	High sensitivity for mRNA	Doesn't reflect protein	Use multiple reference genes
  Proteomics	Unbiased detection	Complex sample preparation	Include spike-in standards
  Microscopy	Spatial information	Subjective quantification	Apply automated image analysis

• Integrated data interpretation:

  • Develop mathematical models incorporating both datasets

  • Consider biological context and known regulatory mechanisms

  • Design validation experiments targeting specific hypotheses
    This systematic approach helps researchers develop coherent explanations for apparent contradictions between transcript and protein levels.

What strategies can researchers employ to minimize background and non-specific binding when using AGD15 Antibody in Western blot applications?

To optimize Western blot performance with AGD15 Antibody, implement these methodological solutions:

• Sample preparation optimization:

  • Include appropriate protease inhibitors to prevent target degradation

  • Use freshly prepared samples whenever possible

  • Determine optimal protein loading amount (typically 10-50 μg per lane)

• Blocking protocol refinement:

  • Test different blocking agents (5% non-fat milk, 3-5% BSA, commercial blockers)

  • Optimize blocking time (1-3 hours at room temperature)

  • Consider adding 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody incubation parameters:

  • Determine optimal antibody dilution through titration (typically 1:500-1:2000)

  • Transition from overnight 4°C to 2-hour room temperature incubation if background persists

  • Add 0.05-0.1% Tween-20 to antibody diluent

• Washing optimization:

  • Increase washing stringency (more wash steps, longer duration)

  • Adjust salt concentration in wash buffers (150-500 mM NaCl)

  • Use fresh wash buffers for each experiment

• Detection system selection:

  • Choose appropriate secondary antibody with minimal cross-reactivity

  • Optimize secondary antibody concentration

  • Consider alternative detection methods (chemiluminescence, fluorescence)
    These approaches follow standard protocols for monoclonal antibody optimization in Western blot applications as outlined in antibody development guidelines .

How should researchers address epitope masking issues when using AGD15 Antibody for detecting native protein complexes?

When investigating native protein complexes with AGD15 Antibody, epitope masking can be addressed through these methodological approaches:

• Sample preparation strategies:

  • Compare denaturing versus non-denaturing conditions

  • Test mild detergents that preserve protein interactions while enabling epitope access

  • Implement gentle lysis techniques to maintain complex integrity

• Epitope accessibility methods:

  • Perform limited proteolysis to expose hidden epitopes

  • Test various antigen retrieval techniques (heat, pH, enzymatic)

  • Consider multiple antibodies targeting different epitopes

• Alternative detection approaches:

  • Use proximity ligation assays to detect proteins in close spatial proximity

  • Implement crosslinking strategies prior to complex disruption

  • Apply super-resolution microscopy techniques for in situ detection

• Controls and validation:

  • Include denatured samples as positive controls for epitope accessibility

  • Use tagged versions of the target protein as reference points

  • Perform reciprocal co-immunoprecipitation experiments

• Experimental design optimization:

  • Compare results across multiple experimental conditions

  • Document the effects of different buffer compositions

  • Consider the temporal dynamics of complex formation
    This comprehensive approach helps distinguish true biological phenomena from technical artifacts when studying protein complexes containing AGD15.

How can AGD15 Antibody be utilized in studying GTPase regulatory networks in cellular signaling pathways?

AGD15 Antibody provides valuable insights into GTPase regulatory networks through these methodological applications:

What are the methodological considerations when using AGD15 Antibody in tissue-specific expression studies?

For tissue-specific expression studies using AGD15 Antibody, researchers should implement these methodological strategies:

• Tissue preparation optimization:

  • Adjust fixation protocols based on tissue type (duration, fixative composition)

  • Optimize sectioning techniques to preserve tissue architecture

  • Implement appropriate antigen retrieval methods for each tissue type

• Background reduction strategies:

  • Block endogenous peroxidase activity for IHC applications

  • Use tissue-specific blocking agents to reduce non-specific binding

  • Include appropriate negative controls for each tissue type

• Signal detection calibration:

  • Determine optimal antibody concentration for each tissue

  • Adjust incubation times based on tissue penetration requirements

  • Select detection systems with appropriate sensitivity for expected expression levels

• Quantification approaches:

  • Develop tissue-specific scoring systems

  • Implement digital image analysis for objective quantification

  • Use multi-parameter analysis to correlate with other markers

• Validation with complementary techniques:

  • Compare immunohistochemistry results with Western blot data

  • Correlate with in situ hybridization for mRNA localization

  • Validate with tissue samples from genetic models with altered expression This comprehensive approach ensures reliable detection of tissue-specific expression patterns while minimizing artifacts and misinterpretation of results.