CAT9 Antibody

What is CAT9 and why are antibodies against it important for research?

CAT9 (cationic amino acid transporter 9) is a tonoplast-localized membrane protein involved in amino acid transport across the vacuolar membrane. In plants such as tomato, SlCAT9 functions as a Glu/Asp/GABA exchanger that influences amino acid accumulation during fruit development. Antibodies against CAT9 are critical for studying its localization, expression levels, and function in plant cellular processes .

How is the anti-CAT9 antibody typically generated?

The anti-CAT9 antibody described in the literature was raised in rabbits against a synthetic peptide (SSALRSKPLASPSET) corresponding to an immunogenic region of SlCAT9. This peptide-based approach allows for the generation of specific antibodies against the target protein. The antibody production was performed as a service by specialized companies such as Eurogentec Ltd .

What are the main applications of CAT9 antibodies in plant research?

CAT9 antibodies are primarily used for:

• Western blotting to detect and quantify CAT9 protein levels in plant tissue extracts

• Immunolocalization studies to confirm the tonoplast localization of CAT9

• Monitoring changes in CAT9 expression during plant development (particularly fruit ripening)

• Validating transgenic lines overexpressing or knocking down CAT9

What is the recommended protocol for using anti-CAT9 antibody in Western blotting?

For Western blotting using anti-CAT9 antibody:

• Extract proteins from plant tissues (e.g., fruit at different ripening stages)

• Separate proteins via SDS-PAGE (typically 10-12% gels)

• Transfer to PVDF membrane

• Block with appropriate blocking buffer (typically 5% non-fat milk)

• Incubate with affinity-purified anti-CAT9 antibody (optimal dilution should be determined empirically, but typically 1:1000)

• Wash thoroughly with TBST

• Incubate with secondary antibody (typically anti-rabbit IgG conjugated to HRP)

• Develop using chemiluminescence detection
  The CAT9 protein should appear as a band around 50 kDa .

How can I validate the specificity of my CAT9 antibody?

Validating antibody specificity is crucial before experimental use. For CAT9 antibody, consider:

• Pre-immune serum control: Compare with pre-immune serum which should not detect any proteins in the extracts

• Peptide competition assay: Pre-incubate antibody with excess immunizing peptide, which should abolish specific signals

• Genetic validation: Test antibodies on CAT9 knockout/knockdown lines, which should show reduced or absent signal

• Heterologous expression: Test on tissues overexpressing tagged CAT9 to confirm co-localization with the tag

• Western blot molecular weight verification: Confirm the band appears at the expected molecular weight (~50 kDa)

What protocols are effective for immunolocalization of CAT9 in plant tissues?

For immunolocalization of CAT9:

• Fix tissue samples in 4% paraformaldehyde

• Embed in appropriate medium (paraffin or resin)

• Section tissues (5-10 μm thickness)

• Perform antigen retrieval if necessary

• Block with BSA or normal serum

• Incubate with anti-CAT9 antibody (1:100-1:500 dilution)

• Wash thoroughly

• Incubate with fluorescently-labeled secondary antibody

• Counterstain nuclei if desired (e.g., with DAPI)

• Mount and visualize using confocal microscopy
  Expected result: CAT9 immunofluorescence should localize to the tonoplast membrane, appearing as a membrane pattern distinct from the cell periphery with transverse strands of intracellular fluorescence representing trans-vacuolar strands .

What are common issues when using CAT9 antibodies and how can they be resolved?

Common issues with CAT9 antibodies include:

How should CAT9 antibodies be stored and handled to maintain optimal activity?

For maximum longevity and activity:

• Store concentrated antibody at -20°C in small aliquots to avoid freeze-thaw cycles

• For short-term storage (1-2 weeks), 4°C is acceptable

• Add preservatives such as sodium azide (0.02%) for long-term storage

• Avoid repeated freeze-thaw cycles

• Centrifuge antibody vial before use to collect all liquid

• Some antibody preparations may contain glycerol (typically 50%) or BSA as stabilizers

• Working dilutions should be prepared fresh before use

How can I use CAT9 antibodies to study the regulation of amino acid transport during fruit development?

Advanced studies on CAT9-mediated amino acid transport can employ:

• Developmental profiling: Use Western blotting with anti-CAT9 antibody to quantify CAT9 protein levels across different fruit developmental stages (from green to red fruit)

• Subcellular fractionation: Isolate tonoplast-enriched membrane fractions and measure CAT9 abundance using quantitative immunoblotting

• Co-immunoprecipitation: Use anti-CAT9 antibodies to identify potential interacting proteins

• Transgenic approaches: Generate plants with altered CAT9 expression and use antibodies to confirm protein levels

• Transport assays: Isolate tonoplast vesicles and perform transport assays with radiolabeled substrates, correlating transport activity with CAT9 protein levels determined by immunoblotting
  Studies have shown that SlCAT9 increases from approximately 0.02% of tonoplast protein at mature green stage to 0.12% in red fruit, correlating with changes in Glu/Asp/GABA transport .

What methods can be used to improve the specificity of CAT9 antibodies for demanding applications?

For enhanced specificity in challenging applications:

• Affinity purification: Purify antibodies against the immunizing peptide using affinity chromatography

• Cross-adsorption: Remove cross-reactive antibodies by pre-incubation with tissue extracts from CAT9 knockout plants

• Epitope mapping: Identify the specific epitope recognized by the antibody using peptide arrays

• CRISPR/Cas9 knockout controls: Generate specific knockout lines as negative controls

• Monoclonal antibody development: Consider developing monoclonal antibodies for increased specificity

• Validation across multiple applications: Ensure antibody works consistently in various techniques (WB, IHC, IP)

How can I adapt CRISPR/Cas9 techniques to validate CAT9 antibody specificity?

CRISPR/Cas9 provides powerful tools for antibody validation:

• Design sgRNAs targeting the CAT9 gene (focusing on early exons)

• Generate CRISPR/Cas9 knockout plants

• Confirm gene editing by sequencing

• Use edited and wild-type tissues side-by-side in antibody validation

• A specific antibody should show significantly reduced or absent signal in knockout tissues

• Alternatively, perform domain-specific knockouts to map the antibody's epitope region
  This approach provides definitive validation of antibody specificity by genetic means rather than relying solely on biochemical methods .

How does anti-CAT9 antibody performance compare to fusion protein approaches for studying CAT9 localization?

Both antibody-based detection and fusion protein approaches have distinct advantages:

What considerations should be taken when designing transport studies correlating CAT9 protein levels with functional activity?

When correlating CAT9 antibody detection with transport function:

• Membrane isolation quality: Ensure high purity of tonoplast membrane preparations (verify using ATPase inhibitor profiles)

• Vesicle integrity: Validate vesicle integrity using proton-pumping assays before transport measurements

• Quantitative Western blotting: Use internal standards for accurate quantification of CAT9 protein

• Transport kinetics: Measure initial rates to avoid complications from equilibration

• Substrate specificity: Test multiple substrates (e.g., Glu, Asp, GABA) to define transport specificity

• Trans-stimulation assays: Design counterexchange experiments to demonstrate exchanger function

• Controls: Include vesicles from plants with altered CAT9 expression levels
  Research has shown that tonoplast vesicles from CAT9-overexpressing plants exhibit higher rates of Glu and GABA transport than wild-type, but only when assayed in counterexchange mode with Glu, Asp, or GABA .

How should researchers design experiments to investigate CAT9 post-translational modifications using antibody-based approaches?

To study CAT9 post-translational modifications:

• 2D gel electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate modified forms

• Phospho-specific antibodies: Generate antibodies against predicted phosphorylation sites

• Immunoprecipitation followed by mass spectrometry: Pull down CAT9 using validated antibodies and analyze by MS

• Treatment studies: Analyze CAT9 modification patterns after treatments affecting protein phosphorylation or other modifications

• Inhibitor studies: Use specific kinase/phosphatase inhibitors to modulate potential modifications

• Mutagenesis: Generate transgenic plants with mutations at putative modification sites and compare antibody recognition patterns
  Note that the molecular weight of CAT9 observed by Western blot (approximately 50 kDa) may vary due to post-translational modifications, which could be studied using these approaches .

How can CAT9 antibodies be integrated with proteomics approaches to study tonoplast transporters?

Integrating antibody detection with proteomics:

• Immunoprecipitation-mass spectrometry (IP-MS): Use anti-CAT9 antibodies to pull down protein complexes for MS analysis

• Quantitative proteomics: Compare tonoplast proteomes across development, correlating changes in CAT9 with other proteins

• Comparative analysis: Use spectral counting or isotope labeling to quantify changes in CAT9 abundance

• Validation: Confirm proteomics findings using Western blotting with anti-CAT9 antibodies

• Protein interaction networks: Build interaction maps based on co-immunoprecipitation data
  In tomato research, quantitative proteomics identified CAT9 as increasing from 0.02% of tonoplast protein at mature green stage to 0.12% in red fruit, making it a candidate protein for the Glu/Asp/GABA exchanger .

What methodological considerations apply when designing antibodies against different domains of the CAT9 protein?

When designing domain-specific antibodies:

• Sequence analysis: Perform multiple sequence alignment to identify unique regions of CAT9

• Hydrophilicity prediction: Select hydrophilic, surface-exposed regions for antibody generation

• Domain targeting: Consider generating antibodies against:

  • N-terminal region (often cytosolic and accessible)

  • C-terminal region (typically cytosolic and accessible)

  • Large loop regions between transmembrane domains

  • Specific functional domains

• Avoid transmembrane regions: These are typically buried in the membrane and make poor antigens

• Epitope conservation: Consider species conservation if cross-reactivity is desired

• Synthetic peptide design: Peptides of 15-20 amino acids generally work well for antibody production
  The successful anti-CAT9 antibody was raised against the peptide SSALRSKPLASPSET, demonstrating the effectiveness of the peptide-based approach .