ATIC Antibody

What is ATIC and what role does it play in cellular metabolism?

ATIC (5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase) is a bifunctional enzyme that catalyzes the last two steps of de novo purine biosynthesis. This 65 kDa protein acts as a transformylase that incorporates a formyl group to the AMP analog AICAR (5-amino-1-(5-phospho-beta-D-ribosyl)imidazole-4-carboxamide) to produce formyl-AICAR (FAICAR), and subsequently catalyzes the cyclization of FAICAR to inosine monophosphate (IMP) .

ATIC has been implicated in several cellular processes beyond purine biosynthesis:

• It can convert thio-AICAR to 6-mercaptopurine ribonucleotide, a purine biosynthesis inhibitor used in leukemia treatment

• It promotes insulin receptor (INSR) autophosphorylation and is involved in INSR internalization

• It has been reported to participate in myeloma and hepatocellular carcinoma progression

How do I select the appropriate ATIC antibody for my research application?

Selecting the right ATIC antibody requires careful consideration of several factors:

• Determine your application: Different ATIC antibodies are validated for specific applications such as Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry (FC), and ELISA. Review the validation data for your specific application .

• Species reactivity: Verify that the antibody recognizes ATIC in your species of interest. Some ATIC antibodies show reactivity across multiple species with high sequence homology, like human, mouse, rat, and other mammals .

• Antibody format: Consider whether a monoclonal or polyclonal antibody better suits your needs:

  • Monoclonal antibodies offer high specificity to a single epitope and batch-to-batch consistency

  • Polyclonal antibodies recognize multiple epitopes and may provide stronger signal

• Validation evidence: Review published literature citations and validation data including Western blot images, ICC/IF images, or flow cytometry data .

• Epitope information: Understanding the binding region (e.g., N-terminal, C-terminal, or middle region) can be important, especially if studying specific domains or protein fragments .

What are the common validation methods for ATIC antibodies?

Proper validation of ATIC antibodies should include multiple approaches:

How should I optimize ATIC antibody dilution for Western blotting?

Optimizing ATIC antibody dilution for Western blotting requires systematic testing:

• Start with manufacturer recommendations: Begin with the suggested dilution range (e.g., 1:2000-1:12000 for some ATIC antibodies) .

• Perform a dilution series: Test 3-4 dilutions across the recommended range using the same sample.

• Control selection: Include positive controls (cell lines known to express ATIC, such as HeLa, HCT116, or Jurkat cells) and negative controls (if available) .

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBS-T for blocking; optimize if background is high.

• Incubation conditions: Test both 1-hour room temperature and overnight 4°C primary antibody incubations to determine optimal conditions.

• Evaluation criteria: Select the dilution that provides:

  • Strong specific bands at the expected molecular weight (65 kDa for ATIC)

  • Minimal background

  • Optimal signal-to-noise ratio

Some ATIC antibodies may require specific optimization based on their formulation and the protein abundance in your samples .

What considerations are important when using ATIC antibodies for immunofluorescence?

Successful immunofluorescence with ATIC antibodies requires attention to several factors:

• Fixation method: Test both paraformaldehyde (4%) and methanol fixation to determine which best preserves ATIC epitopes while maintaining cellular architecture.

• Permeabilization: Since ATIC is primarily cytoplasmic, mild permeabilization with 0.1-0.3% Triton X-100 is typically sufficient.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody to reduce non-specific binding.

• Antibody dilution: Begin with the manufacturer's recommended dilution range (e.g., 1:300-1:1200) and optimize as needed .

• Incubation time and temperature: Test both 1-hour room temperature and overnight 4°C incubations for primary antibody.

• Controls:

  • Positive control: Cell lines with known ATIC expression (e.g., HeLa cells)

  • Negative control: Primary antibody omission

  • If available, siRNA knockdown or CRISPR knockout cells

• Co-staining considerations: When performing double immunostaining, ensure secondary antibodies don't cross-react and choose primary antibodies raised in different species .

• Signal amplification: For low-abundance detection, consider using tyramide signal amplification or other amplification methods.

What are the best approaches for troubleshooting non-specific binding with ATIC antibodies?

Non-specific binding is a common challenge when working with antibodies. Here are systematic approaches to troubleshoot this issue with ATIC antibodies:

• Increase blocking stringency:

  • Extend blocking time to 2 hours or overnight

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Add 0.1-0.3% Triton X-100 to blocking buffer for membrane permeabilization

• Optimize antibody conditions:

  • Further dilute primary antibody

  • Reduce incubation temperature (4°C instead of room temperature)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Try shorter incubation times

• Washing optimization:

  • Increase number of wash steps

  • Extend washing duration

  • Add higher concentrations of detergent to wash buffer

• Validate specificity:

  • Test on known positive and negative control samples

  • Perform peptide competition assay using the immunogen

  • If possible, test on ATIC knockdown/knockout samples

• Secondary antibody considerations:

  • Ensure secondary antibody is compatible with host species of primary antibody

  • Centrifuge secondary antibody before use to remove aggregates

  • Test secondary antibody alone (without primary) to check for non-specific binding

How can ATIC antibodies be employed in studying purine metabolism disorders?

ATIC antibodies can serve as valuable tools in investigating purine metabolism disorders through several advanced applications:

• Expression level analysis: Quantify ATIC protein levels in patient-derived samples compared to healthy controls using Western blotting or immunohistochemistry. Alterations in ATIC expression may correlate with disease severity or progression .

• Enzyme activity correlation: Combine ATIC antibody-based detection with functional assays measuring ATIC enzymatic activity to establish relationships between protein levels and functional outcomes.

• Protein-protein interaction studies:

  • Use ATIC antibodies for co-immunoprecipitation to identify interaction partners

  • Perform proximity ligation assays to visualize and quantify interactions in situ

  • Combine with mass spectrometry to identify novel interaction networks

• Subcellular localization: Employ immunofluorescence with ATIC antibodies to track potential changes in subcellular distribution in disease states.

• Post-translational modification analysis: Use modification-specific antibodies in conjunction with ATIC antibodies to assess how post-translational modifications affect ATIC function in disease contexts.

• Therapeutic response monitoring: Measure changes in ATIC expression or localization following therapeutic interventions targeting purine metabolism.

• Patient stratification: Develop immunohistochemistry-based assays using validated ATIC antibodies to potentially classify patients based on ATIC expression patterns .

What strategies can be used to determine the binding epitope of an ATIC antibody?

Understanding the specific epitope recognized by an ATIC antibody is valuable for interpretation of results and experimental design. Several complementary approaches can be used:

• Peptide array analysis:

  • Create an overlapping peptide array covering the ATIC sequence

  • Probe with the antibody of interest

  • Identify reactive peptides to narrow down binding regions

• Truncation mutants:

  • Generate a series of ATIC truncation constructs

  • Express in mammalian or bacterial systems

  • Perform Western blotting to identify the minimal region required for antibody binding

• Domain swapping:

  • Create chimeric proteins with domains from ATIC and an unrelated protein

  • Test antibody reactivity to identify the domain containing the epitope

• Site-directed mutagenesis:

  • Once a potential epitope region is identified, introduce point mutations

  • Test mutants for altered antibody binding

  • Critical residues will significantly reduce binding when mutated

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare HDX patterns of ATIC alone versus ATIC-antibody complex

  • Regions protected from exchange in the complex indicate binding sites

• X-ray crystallography or cryo-EM: For definitive epitope mapping, determine the structure of the antibody-ATIC complex .

Some ATIC antibodies already have characterized epitopes, such as those targeting aa 379-428 or other specific regions, which can inform experimental design and interpretation .

How can I use ATIC antibodies in multiplexed detection systems?

Multiplexed detection allows simultaneous visualization of ATIC along with other proteins of interest. Advanced strategies include:

• Multicolor immunofluorescence:

  • Select primary antibodies from different host species (e.g., rabbit anti-ATIC and mouse anti-target2)

  • Use spectrally distinct fluorophore-conjugated secondary antibodies

  • Include proper controls to ensure no cross-reactivity between secondary antibodies

  • Consider using directly conjugated primary antibodies for more than 3-4 targets

• Sequential immunostaining:

  • Perform complete staining with first primary and secondary antibodies

  • Use elution buffer to remove antibodies without affecting tissue morphology

  • Repeat staining with subsequent antibody pairs

  • Useful when antibodies are from the same host species

• Mass cytometry (CyTOF):

  • Conjugate ATIC antibodies with distinct metal isotopes

  • Allows for highly multiplexed detection (30+ parameters)

  • Particularly useful for analyzing complex cellular systems

• Proximity ligation assay (PLA):

  • Use ATIC antibody in combination with antibodies against potential interaction partners

  • Generates fluorescent signals only when targets are in close proximity (<40 nm)

  • Enables visualization of protein-protein interactions in situ

• Multiplex immunohistochemistry:

  • Use tyramide signal amplification for sequential staining

  • Each round includes primary antibody, HRP-conjugated secondary, and distinct tyramide-fluorophore

  • Heat-mediated removal of antibodies between rounds

  • Can achieve 6-8 targets on the same tissue section

Optimization of each antibody individually before multiplexing is critical for successful outcomes.

How do I determine if my ATIC antibody has maintained its activity over time?

Antibody degradation can compromise experimental results. These approaches help assess ATIC antibody stability:

• Regular quality control testing:

  • Run Western blot on standard positive control (e.g., HeLa cell lysate)

  • Compare signal intensity and background to results from when antibody was new

  • Document with images for reference

• Appearance assessment:

  • Check for visible precipitation, cloudiness, or color changes

  • Gently mix and observe if any particles remain undissolved

• Stability measurements:

  • If equipped, use dynamic light scattering to check for aggregation

  • Analytical size exclusion chromatography can assess monomer content

• Storage guidelines enforcement:

  • Keep antibodies at recommended temperature (-20°C for most ATIC antibodies)

  • Avoid repeated freeze-thaw cycles by creating small aliquots

  • Some formulations contain 50% glycerol to prevent freezing at -20°C

• Activity monitoring timeline:

  • Test new batch immediately upon receipt to establish baseline

  • Retest after 3, 6, and 12 months to monitor stability

  • Document usage conditions and number of freeze-thaw cycles

• Side-by-side comparison:

  • If possible, maintain a small reference aliquot from initial use

  • Compare performance of current working aliquot with reference sample

What approaches can resolve contradictory results obtained with different ATIC antibodies?

When different ATIC antibodies yield conflicting results, systematic investigation is required:

• Epitope mapping comparison:

  • Determine epitopes recognized by each antibody

  • Differences in accessibility of epitopes in native vs. denatured states may explain discrepancies

  • Post-translational modifications may block certain epitopes

• Validation with orthogonal methods:

  • Use non-antibody detection methods (e.g., mass spectrometry)

  • Employ genetic approaches (siRNA knockdown, CRISPR knockout)

  • Correlate with mRNA expression data

• Sample preparation assessment:

  • Test different lysis buffers, fixation methods, and antigen retrieval protocols

  • Some epitopes may be sensitive to specific preparation conditions

• Antibody format consideration:

  • Compare monoclonal vs. polyclonal antibodies

  • Monoclonals recognize single epitopes which may be lost in certain conditions

  • Polyclonals recognize multiple epitopes but may show more cross-reactivity

• Independent validation:

  • Have different lab members perform experiments blindly

  • Collaborate with other labs to test antibodies under different conditions

• Experimental design optimization:

  • Systematically modify blocking agents, incubation times, temperatures

  • Test different detection systems (chemiluminescence vs. fluorescence)

• Literature reconciliation:

  • Review published studies using these antibodies

  • Contact authors or companies for technical support

How can I validate the specificity of my ATIC antibody in tissues with complex protein mixtures?

Validating antibody specificity in complex tissue samples requires rigorous approaches:

• Genetic validation (gold standard):

  • Test on tissues from ATIC knockout/knockdown models

  • Compare with wild-type tissues processed identically

  • Specific signal should be absent or significantly reduced in knockout samples

• Pre-absorption controls:

  • Pre-incubate antibody with excess purified ATIC protein or immunizing peptide

  • Apply to adjacent tissue sections

  • Specific binding should be blocked in pre-absorbed samples

• Orthogonal method correlation:

  • Compare protein localization with mRNA expression using in situ hybridization

  • Patterns should generally correlate, though post-transcriptional regulation may cause differences

• Multiple antibody verification:

  • Test multiple ATIC antibodies targeting different epitopes

  • Consistent staining patterns increase confidence in specificity

• Western blot correlation:

  • Perform Western blot on tissue lysates from the same source

  • Confirm single band at expected molecular weight (65 kDa)

  • Compare relative expression levels across tissues with IHC staining intensity

• Cell type-specific markers:

  • Co-stain with established cell type markers

  • Confirm expected expression pattern based on known ATIC biology

  • Unexpected localization patterns warrant further investigation

How can machine learning approaches improve ATIC antibody selection and prediction of binding properties?

Machine learning is revolutionizing antibody research, including applications relevant to ATIC antibodies:

• Binding prediction models:

  • Library-on-library approaches can screen many antibodies against many antigens

  • Machine learning models analyze relationships between antibodies and antigens

  • Models can predict binding even for antibodies and antigens not in training data

  • Active learning strategies can reduce experimental data needed by 35%

• Epitope prediction:

  • Algorithms can predict likely binding epitopes based on protein structure and antibody sequences

  • Helps guide experimental design for epitope mapping

  • Can predict cross-reactivity with similar proteins

• Developability prediction:

  • Machine learning models assess physicochemical properties to predict antibody developability

  • Early screening enables elimination of antibodies with suboptimal properties

  • Predictive tools can identify potential aggregation risks in antibodies

• Application-specific performance prediction:

  • Models can predict which ATIC antibodies will perform best in specific applications (WB, IHC, IF)

  • Reduces time spent on empirical testing of multiple antibodies

• Future developments:

  • Integration of structural information with binding data

  • Models accounting for post-translational modifications

  • Transfer learning approaches that leverage data from well-characterized antibodies to improve predictions for new ones

What role do ATIC antibodies play in understanding cancer metabolism and potential therapeutic approaches?

ATIC antibodies serve as critical tools in cancer metabolism research:

• Metabolic pathway analysis:

  • ATIC is upregulated in several cancers including myeloma and hepatocellular carcinoma

  • Antibodies enable quantitative assessment of ATIC expression across cancer types

  • Correlation of expression with patient outcomes provides prognostic insights

• Therapeutic target validation:

  • ATIC can convert thio-AICAR to 6-mercaptopurine ribonucleotide, an anti-leukemia agent

  • Antibodies help validate ATIC as a drug target through expression and functional studies

  • Immunoprecipitation with ATIC antibodies followed by activity assays can assess drug effects on enzymatic function

• Resistance mechanism investigation:

  • Changes in ATIC expression may contribute to therapy resistance

  • Antibodies allow monitoring of expression changes during treatment

  • Co-expression studies with other metabolic enzymes reveal compensatory pathways

• Antibody-drug conjugates (ADCs):

  • If ATIC is surface-expressed in certain cancers, it could be targeted with ADCs

  • ADCs deliver anticancer agents directly to tumor cells expressing the target

  • ATIC antibodies would be the targeting component in such therapies

• Diagnostic applications:

  • ATIC antibodies enable development of diagnostic tests for cancers with altered ATIC expression

  • Immunohistochemistry panels including ATIC may improve cancer classification

  • Liquid biopsy approaches might detect ATIC in circulating tumor cells

• Combination therapy development:

  • ATIC inhibition may sensitize cancers to other therapies

  • Antibodies help assess synergistic effects on metabolic pathways

  • Expression analysis guides rational combination strategies

How can advanced structural biology techniques enhance our understanding of ATIC antibody binding mechanisms?

Advanced structural techniques provide unprecedented insights into antibody-antigen interactions:

• Single-particle analysis techniques:

  • Mass photometry and charge-detection mass spectrometry enable measurement of full IgG binding to target proteins

  • These techniques reveal that antibodies often bind at stoichiometries lower than expected from symmetry

  • Understanding actual binding stoichiometries is critical for interpreting biological effects

• Cryo-electron microscopy (cryo-EM):

  • Provides high-resolution structures of antibody-antigen complexes

  • Reveals conformational epitopes not identifiable through sequence analysis

  • Helps understand steric constraints in complex formation

  • Can visualize structural changes induced by antibody binding

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps regions of ATIC that become protected upon antibody binding

  • Identifies conformational changes in regions distant from binding site

  • Provides dynamic information not available from static structures

• Surface plasmon resonance (SPR) and bio-layer interferometry (BLI):

  • Measures binding kinetics and affinity between ATIC and antibodies

  • Reveals how subtle changes in antibody sequence affect binding properties

  • Enables comparison of different antibodies targeting the same epitope

• X-ray crystallography:

  • Provides atomic-level details of antibody-ATIC complexes

  • Identifies specific amino acid contacts at the binding interface

  • Guides rational antibody engineering efforts

• Molecular dynamics simulations:

  • Leverages structural data to model dynamic interactions

  • Predicts effects of mutations on binding stability

  • Explores conformational changes not captured in static structures