ANTR5 Antibody

What is ANTR5 Antibody and what is its target specificity?

ANTR5 Antibody is a polyclonal antibody that specifically targets and binds to ANTR5 protein in Arabidopsis thaliana (Mouse-ear cress). This antibody is generated by immunizing rabbits with recombinant Arabidopsis thaliana ANTR5 protein as the immunogen . As a research tool, ANTR5 Antibody enables detection, localization, and functional analysis of ANTR5 protein in experimental systems.

The antibody specifically recognizes the native ANTR5 protein (Uniprot No. Q9FKV1) and is purified using antigen affinity methods to ensure high specificity for the target protein . This antibody is designed for research applications only and should not be used in diagnostic or therapeutic procedures.

What applications is ANTR5 Antibody validated for?

ANTR5 Antibody has been validated for several experimental applications, primarily:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

These validated applications make the antibody suitable for quantitative protein detection (ELISA) and qualitative analysis of protein expression, molecular weight determination, and post-translational modifications (Western Blot). While these are the validated applications, researchers might explore additional applications after performing their own validation studies.

What are the proper storage and handling protocols for ANTR5 Antibody?

For optimal performance and stability of ANTR5 Antibody, follow these storage and handling recommendations:

• Upon receipt, store the antibody at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as this can degrade antibody quality

• The antibody is provided in liquid form with a preservative buffer containing 0.03% Proclin 300 and 50% Glycerol in 0.01M PBS, pH 7.4

• Working aliquots should be prepared to minimize freeze-thaw cycles

• When handling the antibody, use sterile pipette tips and sterile microcentrifuge tubes

• Wear gloves to prevent contamination and protein degradation from skin contact

Similar to general antibody handling practices, maintaining cold chain conditions during transportation and avoiding prolonged exposure to room temperature will help preserve antibody activity and specificity .

How can I optimize Western blot protocols for ANTR5 Antibody?

For optimal Western blot results with ANTR5 Antibody, consider the following methodological approaches:

• Sample Preparation:

  • Extract plant proteins using an appropriate buffer that preserves protein integrity

  • Add protease inhibitors to prevent protein degradation

  • Denature proteins completely using standard SDS-PAGE sample buffer with heat treatment

• Blocking and Antibody Incubation:

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

  • Start with a 1:200 dilution of ANTR5 Antibody as recommended , then optimize based on results

  • Incubate primary antibody at 4°C overnight for better sensitivity and specificity

  • Use TBS-T with 0.1% Tween-20 for washing steps

• Detection System:

  • Choose an appropriate HRP-conjugated secondary anti-rabbit antibody

  • Consider using enhanced chemiluminescence (ECL) for detection

  • For quantitative analysis, consider digital imaging systems with exposure optimization

• Validation Controls:

  • Include positive control (Arabidopsis thaliana extracts)

  • Include negative control (non-plant sample)

  • Perform a peptide competition assay by pre-incubating the antibody with its immunizing peptide

This approach mirrors standard protocols used for other plant antibodies, similar to the Western blot analysis shown for other antibodies in the literature .

What controls should be included when working with ANTR5 Antibody?

Including appropriate controls is crucial for interpreting results with ANTR5 Antibody:

• Positive Controls:

  • Arabidopsis thaliana wild-type tissues known to express ANTR5

  • Recombinant ANTR5 protein expressed in a heterologous system

  • Previously validated samples with confirmed ANTR5 expression

• Negative Controls:

  • Tissues from ANTR5 knockout/knockdown plants (if available)

  • Non-plant tissues or samples from organisms lacking ANTR5 homologs

  • Secondary antibody-only controls to assess non-specific binding

• Peptide Competition/Blocking Controls:

  • Pre-incubate ANTR5 Antibody with excess immunizing peptide to confirm specificity

  • This approach is similar to the blocking peptide validation shown for other antibodies

• Loading Controls:

  • Include detection of housekeeping proteins (e.g., actin, tubulin) to normalize expression levels

  • Use total protein staining methods (e.g., Ponceau S) to verify equal loading

These controls help distinguish specific signals from non-specific background and validate that observed signals truly represent ANTR5 protein, similar to validation methods used for other antibodies in research .

How does sample preparation affect ANTR5 detection in plant tissues?

Sample preparation significantly impacts the success of ANTR5 detection in plant tissues:

• Extraction Buffers:

  • Use buffer systems that maintain protein stability (e.g., Tris-HCl pH 7.5-8.0 with 150mM NaCl)

  • Include detergents appropriate for membrane proteins if ANTR5 has membrane associations

  • Add protease inhibitors to prevent degradation during extraction

• Tissue Processing:

  • Fresh tissue yields better results than stored samples

  • Flash-freezing in liquid nitrogen followed by mechanical disruption (grinding) preserves protein integrity

  • Centrifugation steps should be optimized to separate cellular debris while retaining the protein fraction of interest

• Fixation for Immunohistochemistry:

  • If using ANTR5 Antibody for tissue localization, fixation methods impact epitope accessibility

  • Test both aldehyde-based (e.g., 4% paraformaldehyde) and alcohol-based fixatives

  • Consider antigen retrieval methods if fixation reduces antibody binding

• Protein Denaturation:

  • For Western blotting, complete denaturation in SDS-containing buffer with heating (95-100°C for 5 minutes)

  • For native conditions (e.g., immunoprecipitation), use milder non-ionic detergents and avoid heating

Optimizing these parameters will improve detection sensitivity and specificity, as demonstrated in protocols for other plant antibodies and protein detection methods.

How can I troubleshoot weak or non-specific signals with ANTR5 Antibody?

When encountering weak signals or non-specific binding with ANTR5 Antibody, consider these troubleshooting approaches:

• For Weak Signals:

  Parameter	Adjustment	Rationale
  Antibody concentration	Increase from 1:200 to 1:100 or 1:50	Higher concentration may improve detection of low-abundance targets
  Incubation time	Extend to overnight at 4°C	Allows more complete antibody-antigen binding
  Protein loading	Increase amount loaded	Ensures detectable target protein levels
  Detection system	Switch to more sensitive ECL substrate	Amplifies signal from low-abundance proteins
  Sample preparation	Enrich for target protein fraction	Concentrates the protein of interest

• For Non-specific Signals:

  Parameter	Adjustment	Rationale
  Blocking conditions	Increase blocking time/concentration	Reduces non-specific binding sites
  Wash stringency	Increase wash duration/detergent	Removes weakly bound antibody
  Antibody dilution	Increase dilution (1:300-1:500)	Reduces excess antibody contributing to background
  Blocking agent	Try different blockers (milk vs. BSA)	Different blockers perform better with different antibodies
  Pre-adsorption	Pre-incubate antibody with non-target tissues	Removes antibodies binding to cross-reactive epitopes

• Additional Considerations:

  • Verify protein extraction efficiency with total protein stains

  • Check for protein degradation with fresh samples and additional protease inhibitors

  • Consider epitope masking that might occur due to protein folding or interactions

  • Test alternative membrane types for Western blotting (PVDF vs. nitrocellulose)

This systematic approach to optimization follows standard troubleshooting practices for immunoassays and has been effective for other antibodies in research settings .

How can I validate cross-reactivity with other plant species beyond Arabidopsis thaliana?

Validating ANTR5 Antibody cross-reactivity with other plant species requires a strategic approach:

• Sequence Homology Analysis:

  • Perform bioinformatic analysis of the immunogen sequence (residues used to generate the antibody) across potential target species

  • Higher sequence homology suggests higher likelihood of cross-reactivity

  • Focus on conserved domains or regions within the ANTR5 protein

• Western Blot Validation:

  • Test protein extracts from candidate plant species in parallel with Arabidopsis controls

  • Begin with closely related species in the Brassicaceae family

  • Verify that detected bands match the expected molecular weight of ANTR5 homologs

  • Confirm specificity with peptide competition assays

• Immunoprecipitation Followed by Mass Spectrometry:

  • Use ANTR5 Antibody to immunoprecipitate potential cross-reactive proteins

  • Identify captured proteins by mass spectrometry

  • Compare identified proteins to known ANTR5 homologs

• Validation Using Transgenic Systems:

  • Express the ANTR5 homolog from the target species in a heterologous system

  • Test antibody binding to the expressed protein

  • Compare binding efficiency to that of Arabidopsis ANTR5

This methodical approach is similar to the validation strategies used for other antibodies across species, such as those demonstrated for the Nav1.5 antibody across different mammalian systems .

What quantitative approaches can be used with ANTR5 Antibody for expression analysis?

For quantitative analysis of ANTR5 expression using ANTR5 Antibody, consider these methodological approaches:

• Quantitative Western Blotting:

  • Use digital imaging systems with linear detection range

  • Include calibration standards with known concentrations

  • Normalize to loading controls (housekeeping proteins or total protein stains)

  • Analyze band intensity using appropriate software (ImageJ, etc.)

  Sample preparation	Detection method	Normalization	Software analysis
  Standardized protein extraction	Chemiluminescence with digital capture	Total protein normalization (Ponceau S)	ImageJ densitometry
  Bradford-quantified lysates	Near-infrared fluorescence detection	Housekeeping protein controls	Proprietary analysis software
  Serial dilutions for standard curve	Low-fluorescence PVDF membranes	Recombinant protein standards	Statistical validation (ANOVA)

• Quantitative ELISA:

  • Develop a sandwich ELISA using ANTR5 Antibody

  • Generate standard curves with recombinant ANTR5 protein

  • Ensure samples fall within the linear range of detection

  • Calculate absolute concentrations based on standard curves

• Immunohistochemistry with Digital Image Analysis:

  • Use consistent staining protocols and imaging parameters

  • Apply digital image analysis to quantify signal intensity

  • Compare relative expression levels across tissues or treatments

  • Include internal reference standards in each experiment

• Flow Cytometry for Protoplasts:

  • Label plant protoplasts with ANTR5 Antibody

  • Quantify fluorescence intensity per cell

  • Analyze population distributions and mean fluorescence intensity

These quantitative approaches mirror those used in antibody-based quantification of other proteins, incorporating standardization and calibration methods to ensure reliable comparative analyses .

How can ANTR5 Antibody be used to study protein-protein interactions?

ANTR5 Antibody can be employed in several methodologies to investigate protein-protein interactions:

• Co-Immunoprecipitation (Co-IP):

  • Use ANTR5 Antibody to precipitate ANTR5 protein and associated complexes

  • Identify interacting partners through subsequent Western blotting or mass spectrometry

  • Compare results under different physiological conditions to identify dynamic interactions

  • Include appropriate controls (IgG control, lysates without antibody) to confirm specificity

• Proximity Ligation Assay (PLA):

  • Combine ANTR5 Antibody with antibodies against potential interacting partners

  • Visualize and quantify interactions at the subcellular level

  • This technique detects proteins in close proximity (<40 nm), suggesting direct interaction

• Pull-down Assays with Validation:

  • Use ANTR5 Antibody to validate interactions identified through other methods

  • Confirm specificity of interactions by competition with excess antigen

  • Test interactions under different buffer conditions to assess strength and specificity

• Immunofluorescence Co-localization:

  • Perform double immunolabeling with ANTR5 Antibody and antibodies against potential partners

  • Quantify co-localization using appropriate statistical methods

  • Use high-resolution microscopy (confocal, STED) for detailed spatial analysis

These approaches are similar to interaction studies performed with other antibodies, such as those described for cardiac sodium channels in the search results , adapted to plant cell biology contexts.

What is the role of epitope mapping in understanding ANTR5 Antibody functionality?

Understanding the epitope recognized by ANTR5 Antibody provides valuable insights into antibody functionality:

• Importance of Epitope Mapping:

  • Reveals the specific region of ANTR5 protein recognized by the antibody

  • Helps predict potential cross-reactivity with related proteins

  • Informs experimental design when studying protein domains or post-translational modifications

  • Assists in interpreting negative results (epitope might be masked in certain conditions)

• Mapping Techniques:

  • Peptide array analysis using overlapping peptides from ANTR5 sequence

  • Mutagenesis studies to identify critical binding residues

  • Hydrogen-deuterium exchange mass spectrometry to identify protected regions

  • X-ray crystallography of antibody-antigen complexes (for detailed structural analysis)

• Practical Applications of Epitope Knowledge:

  • Evaluate accessibility of the epitope in different experimental conditions

  • Design blocking peptides for specificity controls

  • Predict effects of protein conformational changes on antibody binding

  • Understand potential limitations in certain applications (e.g., non-denaturing conditions)

Epitope mapping approaches similar to those used for clinical antibody development can be adapted for research antibodies like ANTR5 Antibody, as demonstrated in antibody characterization studies .

How might post-translational modifications affect ANTR5 Antibody binding?

Post-translational modifications (PTMs) can significantly impact ANTR5 Antibody binding, with important experimental implications:

• Potential Effects of PTMs on Antibody Recognition:

  • Phosphorylation, glycosylation, or other modifications may directly block antibody binding sites

  • PTMs can induce conformational changes that indirectly affect epitope accessibility

  • Some PTMs might create new epitopes not recognized by the antibody

  • Certain modifications might enhance antibody binding through increased epitope exposure

• Experimental Approaches to Address PTM Interference:

  • Compare antibody binding under conditions that preserve or remove PTMs

  • Use phosphatase or glycosidase treatments to remove specific modifications

  • Combine ANTR5 Antibody with modification-specific antibodies in sequential or parallel detection

  • Develop validation assays using recombinant ANTR5 with and without specific modifications

• Interpreting Results with PTM Considerations:

  • Variable detection across samples may reflect differential modification states

  • Absence of signal may indicate modification-based epitope masking rather than absence of protein

  • Multiple bands on Western blots might represent differently modified forms of ANTR5

This consideration of PTM effects on antibody binding is important for accurate interpretation of experimental results, similar to methodological considerations in protein detection systems for other research antibodies .

How does ANTR5 Antibody performance compare with genetic tagging approaches?

Researchers should consider the relative advantages of antibody-based detection versus genetic tagging when studying ANTR5:

Complementary approaches combining antibody detection of native ANTR5 with tagged versions can provide validation and extended experimental capabilities, similar to complementary approaches used in other protein studies .

What are the best practices for validating ANTR5 Antibody specificity in knockout/knockdown studies?

Rigorous validation of ANTR5 Antibody using genetic knockout or knockdown approaches follows these best practices:

• Experimental Design for Validation:

  • Compare wild-type plants with confirmed ANTR5 knockout/knockdown lines

  • Include heterozygous plants as intermediate expression controls

  • Analyze multiple independent knockout/knockdown lines to rule out off-target effects

  • Process all samples identically to eliminate technical variables

• Critical Controls:

  • Include loading controls to normalize protein amounts

  • Process wild-type and knockout samples in parallel

  • Include positive controls for antibody functionality

  • Test multiple tissues where ANTR5 is expressed at different levels

• Analysis and Interpretation:

  • Complete absence of signal in knockout lines confirms high specificity

  • Reduced signal proportional to knockdown efficiency supports specificity

  • Persistent bands in verified knockout lines suggest cross-reactivity

  • Quantify signal reduction relative to mRNA reduction in knockdown lines

• Addressing Partial Validation Results:

  • If residual signal exists in knockout lines, perform peptide competition assays

  • Consider mass spectrometry to identify proteins detected in knockout samples

  • Evaluate potential isoforms or homologs that might be detected

This rigorous validation approach follows standard practices in antibody characterization as demonstrated in similar validation studies for other research antibodies .