ANTR1 Antibody

What is ANTR1 and how does it differ from ANTXR1?

There appears to be some confusion in the literature between two distinct proteins with similar abbreviations. ANTR1 (Arabidopsis Nucleotide/Phosphate Transporter 1) is a membrane protein involved in phosphate transport, primarily studied in Arabidopsis thaliana. It belongs to the MFS (Major Facilitator Superfamily) of transporters .

In contrast, ANTXR1 (Anthrax Toxin Receptor 1), also known as ATR and TEM8, is a type I transmembrane protein that serves as a docking protein or receptor for Bacillus anthracis toxin. ANTXR1 is a tumor-specific endothelial marker implicated in colorectal cancer and is highly expressed in tumor endothelial cells .

These proteins have entirely different functions and cellular localizations, with ANTR1 containing 12 transmembrane segments and functioning in phosphate transport , while ANTXR1 has a calculated molecular weight of 63 kDa and interacts with anthrax toxin . This distinction is critical when selecting appropriate antibodies for specific research applications.

What are the key characteristics of commercially available ANTR1/ANTXR1 antibodies?

Commercial antibodies against these proteins typically have the following characteristics:

• Host/Isotype: Most are rabbit polyclonal IgG antibodies, though some monoclonal options may be available .

• Immunogen: Antibodies are typically raised against specific peptide sequences, particularly from C-terminal or internal regions .

• Reactivity: Many antibodies show reactivity with multiple species, including human, mouse, and rat samples .

• Applications: Common applications include Western Blotting (WB), Immunofluorescence (IF), and Enzyme Immunoassays (EIA) .

• Specificity: Some antibodies are designed to recognize specific regions or isoforms of the protein. For example, some ANTXR1 antibodies recognize only isoform 1 .

• Form and Storage: Typically supplied in liquid form with storage buffers containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, recommended to be stored at -20°C .

How should researchers validate ANTR1/ANTXR1 antibodies before experimental use?

Proper validation is crucial for reliable results. A methodological approach includes:

• Western Blot Validation: Confirm the antibody detects a protein of the expected molecular weight (e.g., 63 kDa for ANTXR1 or 56 kDa for ANTR1 ).

• Negative Controls: Test the antibody in systems where the target protein is not expressed. For example, when examining ANTR1 expression in E. coli upon IPTG induction, control cells showed no signal with anti-FLAG antibody detection .

• Multiple Antibody Comparison: Where possible, compare results with multiple antibodies targeting different epitopes of the same protein. For instance, researchers have used both anti-FLAG and ANTR1-specific antibodies to verify expression patterns .

• Peptide Competition: Pre-incubate the antibody with the immunizing peptide to verify that the signal disappears in your experimental system.

• Knockdown/Knockout Controls: If available, compare antibody signal in wild-type versus samples where the target protein has been genetically reduced or eliminated.

• Cross-reactivity Testing: Test antibody specificity across multiple species or closely related proteins if your research requires such discrimination.

What optimal conditions should be used for storage and handling of these antibodies?

To maintain antibody functionality:

• Storage Temperature: Store at -20°C for long-term stability. Most antibodies remain stable for one year after shipment when properly stored .

• Buffer Composition: The typical storage buffer contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles .

• Aliquoting Considerations: For antibodies stored at -20°C, aliquoting is generally unnecessary, though for frequently used antibodies, it may help minimize freeze-thaw cycles .

• Stabilizing Additives: Some smaller volume preparations (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizer .

• Working Dilutions: Prepare working dilutions immediately before use and avoid storing diluted antibody for extended periods.

• Contamination Prevention: Use sterile techniques and avoid microbial contamination, which can degrade antibody performance.

How do researchers optimize ANTR1/ANTXR1 antibodies for specific experimental applications?

Application-specific optimization requires methodological adjustments:

• Western Blotting: Optimization typically involves testing different antibody dilutions, blocking reagents, and detection systems. For membrane proteins like ANTR1, sample preparation is crucial - ensure complete solubilization of membrane fractions before electrophoresis .

• Immunofluorescence: Fixation method can significantly impact epitope accessibility. Test both paraformaldehyde and methanol fixation, particularly for membrane proteins, which may require permeabilization optimization with detergents like Triton X-100 or saponin.

• ELISA Applications: ANTXR1 antibodies have been successfully used in ELISA applications with human, mouse, and rat samples . Optimization involves testing different coating buffers, blocking reagents, and antibody incubation times.

• Quantitative Analysis: For quantitative Western blot analyses, researchers have successfully used densitometry to compare expression levels of wild-type and mutant proteins, with expression levels ranging from 80% to 200% relative to wild-type .

• Cross-Validation: Where possible, use orthogonal detection methods. For instance, researchers have used both anti-FLAG antibody and ANTR1-specific antibody to verify protein expression patterns .

What role do specific amino acid residues play in ANTR1 function and antibody recognition?

Research on conserved amino acid residues provides valuable insights:

• Functional Classification: Conserved residues in ANTR1 have been classified into different functional categories, including binding/translocation-related, exposed, closed, or other roles . The 25 most conserved residues across ANTR1 homologues have been systematically analyzed, as shown in this table:

• Mutational Analysis: Site-directed mutagenesis of key residues (R120, S124, R201, R228, and D382) has revealed their importance for protein function. Notably, mutations in R228 and D382 dramatically increased protein expression levels (160-200% of wild-type) .

• Antibody Epitope Considerations: Exposed residues like R228 and D382 represent potential epitopes for antibody recognition. Understanding their conservation and accessibility can guide epitope selection for antibody development .

• Structural Implications: The location of these residues within specific transmembrane segments (TM) helps define the protein's topology, which is crucial for understanding which regions might be accessible to antibodies in different experimental contexts .

How can researchers distinguish between closely related protein isoforms using antibodies?

Isoform-specific detection requires careful methodological approaches:

• Epitope Selection: Target unique sequences that differ between isoforms. For example, some ANTXR1 antibodies specifically recognize only isoform 1 .

• C-terminal Targeting: C-terminal regions often differ between isoforms and make good targets for isoform-specific antibodies. Some commercial antibodies target the C-terminus of ANTXR1 (e.g., amino acids 358-386 or other C-terminal regions) .

• Western Blot Analysis: Different isoforms often have distinct molecular weights that can be resolved by SDS-PAGE. Careful optimization of gel percentage and running conditions can enhance separation.

• Positive Controls: Express recombinant versions of specific isoforms as positive controls to confirm antibody specificity.

• Blocking Peptides: Use isoform-specific peptides in competition assays to confirm antibody specificity.

• Alternative Splicing Awareness: Knowledge of alternative splicing patterns can guide epitope selection. Design antibodies targeting exon junctions unique to specific splice variants.

What methodological approaches are most effective for detecting post-translational modifications of ANTR1/ANTXR1?

Detecting post-translational modifications requires specialized approaches:

• Phosphorylation-Specific Antibodies: Commercial antibodies targeting specific phosphorylation sites, such as pTyr425 and pTyr382 in ANTXR1, are available and can be used to study signaling events .

• Mass Spectrometry: For comprehensive identification of multiple post-translational modifications, immunoprecipitate the protein using a general ANTR1 antibody, then analyze by mass spectrometry.

• Mobility Shift Assays: Some modifications alter protein mobility on SDS-PAGE. Compare the migration patterns of the protein before and after treatment with phosphatases or other modification-removing enzymes.

• 2D Gel Electrophoresis: This approach can separate protein isoforms based on both molecular weight and isoelectric point, which often changes with phosphorylation or other modifications.

• Functional Correlation: Correlate the presence of specific modifications with functional outcomes using transport assays or other functional readouts.

How can computational approaches enhance ANTR1/ANTXR1 antibody design and characterization?

Advanced computational methods offer powerful approaches for antibody research:

• Homology Modeling: Structural models of ANTR1 have been developed using templates like GlpT (PDB ID: 1PW4) despite only 18% sequence identity. These models provide insights into protein structure that can guide epitope selection for antibody development .

• Transmembrane Helix Prediction: Tools in databases like ARAMEMNON apply multiple prediction methods to identify transmembrane segments. Most methods predict 9-11 transmembrane segments for ANTR1, while some predict the 12 TMs that correspond with crystallized MFS members .

• Multiple Sequence Alignment (MSA): MSA of ANTR1 with related proteins helps identify conserved regions that may be functionally important and good antibody targets. Four different MSAs have been used to identify the most conserved residues in ANTR1 .

• Antibody-Glycan Modeling: Similar approaches to those used for glycan-binding antibodies could be adapted for ANTR1/ANTXR1. These methods combine high-throughput screening, site-directed mutagenesis, and saturation transfer difference NMR to define antibody-antigen interactions .

• Molecular Dynamics Simulations: These can refine homology models and predict conformational changes relevant to antibody binding. The AbPredict algorithm combines segments from various antibodies and samples large conformational spaces to generate low-energy models .

What are the challenges in developing highly specific antibodies for functional studies of ANTR1/ANTXR1?

Researchers face several methodological challenges:

• Membrane Protein Topology: With 12 predicted transmembrane segments, many portions of ANTR1 are embedded in the lipid bilayer and inaccessible to antibodies in intact systems . This constrains epitope selection to extracellular/luminal domains or cytoplasmic regions.

• Conformational Epitopes: The native conformation of these membrane proteins presents epitopes that may be difficult to mimic with peptide immunogens, potentially limiting antibody effectiveness.

• Isoform Complexity: Multiple isoforms can complicate antibody development. For example, some ANTXR1 antibodies specifically recognize only isoform 1 , requiring careful epitope selection for isoform discrimination.

• Cross-reactivity Control: Ensuring specificity against closely related proteins requires comprehensive validation against potential cross-reactants.

• Functional Domain Targeting: For antibodies intended to modulate protein function, epitopes must be carefully selected near functional domains without disrupting protein structure.

• Species Cross-reactivity: Developing antibodies that work across species requires targeting highly conserved epitopes, which may limit epitope options.

How can ANTR1/ANTXR1 antibodies be used to investigate structure-function relationships?

Advanced structure-function studies employ several methodological approaches:

• Mutant Analysis: Combining site-directed mutagenesis with antibody detection allows researchers to investigate how mutations affect protein expression and function. For ANTR1, mutations in key residues (R120, S124, R201, R228, D382) have been studied for their effects on phosphate transport and sodium dependency .

• Epitope Mapping: Fine mapping of antibody epitopes can provide structural insights, especially when combined with functional assays.

• Accessibility Studies: Antibodies targeting different domains can probe protein topology and conformational changes by testing accessibility under different conditions.

• Co-immunoprecipitation: ANTR1/ANTXR1 antibodies can pull down the target protein along with interaction partners, revealing functional protein complexes.

• Inhibitory Antibodies: Antibodies targeting functional domains might inhibit substrate binding or transport, providing insights into mechanistic details.

• Conformational State Detection: Some antibodies preferentially bind specific conformational states, helping to investigate structural changes during transport cycles.

What role do ANTR1/ANTXR1 antibodies play in investigating pathogen-host interactions and disease mechanisms?

These antibodies enable several critical research applications:

• Anthrax Toxin Interactions: ANTXR1 (also known as TEM8) serves as a receptor for Bacillus anthracis toxin. Antibodies against ANTXR1 can be used to study this interaction and potentially block toxin binding .

• Protective Immunity: Studies of vaccinated individuals have used antibodies to evaluate immune responses to anthrax protective antigen (PA). High titers of antibodies to PA follow anthrax vaccine adsorbed (AVA) vaccination, with levels correlating with the number of vaccinations received .

• Population Studies: Antibody-based assays have been used to study vaccination responses across diverse populations, as shown in this demographic table:

• Cancer Research: As a tumor-specific endothelial marker implicated in colorectal cancer, ANTXR1/TEM8 antibodies can be used to study tumor angiogenesis and potentially develop targeted therapies .

• Epitope-Specific Protection: Peptide-specific antibodies enriched from sera of vaccinated individuals have demonstrated in vivo protection against lethal toxin, indicating the potential therapeutic value of targeted antibody responses .