IMPDH2 Antibody Pair

What is IMPDH2 and why is it important in cellular research?

IMPDH2 (Inosine-5'-monophosphate dehydrogenase 2) is a rate-limiting enzyme in the de novo guanine nucleotide biosynthesis pathway. It catalyzes the NAD-dependent oxidation of inosine-5'-monophosphate (IMP) into xanthine-5'-monophosphate (XMP), which is subsequently converted into guanosine-5'-monophosphate (GMP) . The enzyme plays a critical role in maintaining cellular guanine deoxy- and ribonucleotide pools necessary for DNA and RNA synthesis. IMPDH2 is particularly important in rapidly proliferating cells and is upregulated in some neoplasms, suggesting involvement in malignant transformation . Recent studies have also identified IMPDH2 as a potential therapeutic target in conditions requiring control of cell proliferation .

What is an IMPDH2 antibody pair and how does it differ from single antibodies?

An IMPDH2 antibody pair consists of two complementary antibodies designed to work together in detection assays:

• Capture/Primary Antibody: Typically used for immunoprecipitation (IP) or as the capture antibody in ELISA. For example, mouse monoclonal anti-IMPDH2 (300 μg) is commonly used for IP .

• Detection/Secondary Antibody: Used to detect the precipitated protein, often in Western blot (WB) or as the detection antibody in ELISA. For example, rabbit polyclonal anti-IMPDH2 (50 μl) for WB .

Unlike single antibodies, antibody pairs ensure higher specificity by requiring two distinct epitope recognition events, significantly reducing background and cross-reactivity. This dual-antibody approach is particularly valuable for detecting endogenous IMPDH2 in complex biological samples with minimal non-specific binding .

What applications are IMPDH2 antibody pairs most commonly used for?

IMPDH2 antibody pairs are primarily optimized for:

• Immunoprecipitation-Western Blot (IP-WB): For pulling down IMPDH2 from cell lysates and confirming its presence via Western blot

• Sandwich ELISA: For quantitative detection of IMPDH2 in samples using a capture and detection antibody system

• Immunofluorescence (IF): For visualizing IMPDH2 filament formation in cells under various conditions

• Immunohistochemistry (IHC): For detecting IMPDH2 expression in tissue sections, particularly in cancer research

• Flow Cytometry: For analyzing IMPDH2 expression in individual cells, especially in immune cell activation studies

The selection of specific applications depends on experimental goals. For instance, IP-WB combinations are ideal for studying protein-protein interactions, while ELISA is preferred for quantitative analysis of IMPDH2 levels in biological fluids .

How should IMPDH2 antibody pairs be stored and handled to maintain optimal activity?

Proper storage and handling of IMPDH2 antibody pairs is crucial for maintaining their functionality:

Handling recommendations:

• Store reagents of the antibody pair set at -20°C or lower

• Aliquot reconstituted antibodies to avoid repeated freeze-thaw cycles

• Return reagents to -20°C storage immediately after use

• When reconstituting lyophilized antibodies, add the recommended volume of distilled water to yield the appropriate concentration (e.g., adding 0.2 ml to yield 500 μg/ml)

• Allow antibodies to equilibrate to room temperature before opening vials to prevent condensation

Failure to follow these storage guidelines can result in antibody degradation, leading to reduced sensitivity and increased background in experimental applications .

What are the optimal conditions for using IMPDH2 antibody pairs in IP-WB applications?

To achieve optimal results with IMPDH2 antibody pairs in IP-WB applications:

Immunoprecipitation:

• Use mouse monoclonal anti-IMPDH2 for IP at a recommended concentration of 2-5 μg per 500 μg of total protein

• Utilize protein A magnetic beads (e.g., U0007) for efficient capture

• Incubate antibody-sample mixture overnight at 4°C with gentle rotation

• Wash precipitates at least 3-5 times with cold IP buffer to reduce background

Western Blot:

• Use rabbit polyclonal anti-IMPDH2 at a dilution of 1:1000 for detection

• Block membranes with 5% non-fat milk or BSA in TBST for at least 1 hour at room temperature

• Incubate with primary antibody overnight at 4°C

• Wash thoroughly with TBST before adding secondary antibody

• For IMPDH2 detection, look for bands at approximately 55.8 kDa

Quality control considerations:

• Always include positive controls (e.g., IMPDH2 transfected lysates)

• Include negative controls (e.g., IgG from the same species as the IP antibody)

• For challenging samples, consider pre-clearing lysates with protein A beads before IP to reduce non-specific binding

These optimized conditions have been validated through quality control testing for specific IMPDH2 antibody pairs .

How can I optimize IMPDH2 antibody pairs for detecting IMPDH2 filament formation?

IMPDH2 can form filaments (cytoophidia or rods and rings structures) under specific cellular conditions. For optimal detection of these structures:

Immunofluorescence protocol optimization:

• Fixation method: Use 4% paraformaldehyde for 15 minutes at room temperature, as this preserves filament structures better than methanol fixation

• Permeabilization: Use 0.1-0.5% Triton X-100 in PBS for 10 minutes

• Antibody dilution: Use rabbit polyclonal anti-IMPDH2 at 1:100-1:500 dilution for primary detection

• Incubation time: Extend primary antibody incubation to overnight at 4°C for enhanced sensitivity

• Mounting medium: Use anti-fade mounting medium containing DAPI for nuclear counterstaining

Experimental conditions that promote IMPDH2 filament formation:

• Treatment with IMPDH inhibitors such as mycophenolic acid (MPA) at 1-100 μM or ribavirin (RBV) at 500 μM

• ATP concentration as low as 1 μM can induce assembly in vitro

• T-cell activation with mitogens like phytohemagglutinin (PHA) or concanavalin A (ConA)

• Rapidly proliferating cells such as activated lymphocytes or regenerating tissues

The filaments appear as extended structures that can be visualized clearly with confocal microscopy. In regenerating tissues, lower doses of MPA (1-10 μM) can induce abundant superstructures, showing a sensitized environment for IMPDH2 filament formation .

How can IMPDH2 antibody pairs be used to study the relationship between IMPDH2 and cell proliferation in cancer research?

IMPDH2 is frequently upregulated in neoplasms and plays a critical role in cell proliferation. For studying this relationship:

Experimental approach using antibody pairs:

• Comparative expression analysis:

  • Use sandwich ELISA with IMPDH2 antibody pairs to quantify IMPDH2 levels across normal and cancer tissues

  • Correlate expression levels with clinicopathological features using tissue microarrays and IHC scoring systems

• Functional studies:

  • Combine IMPDH2 knockdown (via siRNA) with antibody detection to monitor effects on cell proliferation

  • Use CCK-8 assays to measure cell viability after IMPDH2 modulation

  • Perform colony formation assays followed by IMPDH2 detection to link expression to clonogenic potential

• Cell cycle analysis:

  • Use flow cytometry with PI staining to determine cell cycle phase distribution after IMPDH2 knockdown

  • Correlate with IMPDH2 expression using antibody pairs in parallel samples

This approach enables researchers to establish direct links between IMPDH2 expression levels and cancer cell proliferation, potentially identifying new therapeutic strategies targeting the guanine nucleotide synthesis pathway .

What methods can be used to study IMPDH2 filament assembly and its regulatory mechanisms?

IMPDH2 can form filaments that regulate its activity through conformational changes. To study this process:

Cryo-EM structural analysis:

• Induce IMPDH2 filament assembly in vitro by adding ATP (concentrations as low as 1 μM)

• Create more structurally homogeneous filaments by addition of IMP or NAD

• Use negative stain EM to characterize filament morphology under different conditions

• Apply cryo-EM for high-resolution structural analysis of IMPDH2 filaments in both active and inactive conformations

Regulatory mechanism studies:

• Analyze GTP dose-response of wildtype IMPDH2 versus non-assembling mutants (e.g., Y12A)

• Compare enzyme kinetics between filamentous and non-filamentous forms

• Examine the effects of substrate (IMP) and product (GTP) on filament assembly and disassembly

• Investigate competition between ATP and GTP at the Bateman domains

Conformational state analysis:
Through cryo-EM studies, researchers have established that IMPDH2 filaments exist in distinct conformational states:

• "Bowed" tetramer conformation (inhibited state)

• "Flat" tetramer conformation (active state)

• Extended and compressed filament segments under various ligand conditions

This structural information reveals how filament assembly modulates IMPDH2 activity, making the enzyme less sensitive to feedback inhibition by GTP, particularly under conditions requiring expansion of guanine nucleotide pools .

How can IMPDH2 antibody pairs be used to investigate IMPDH2's role in immune response and T-cell activation?

IMPDH2 plays a crucial role in immune cell activation and proliferation. To investigate this:

T-cell activation studies:

• Ex vivo activation model:

  • Isolate T cells and activate them with mitogens (PHA, ConA) or antibodies to CD3/CD28

  • Use immunofluorescence with IMPDH2 antibody pairs to detect filament formation

  • Quantify the percentage of T cells showing IMPDH2 filaments (40-60% in activated vs. 0-10% in unstimulated T cells)

• In vivo antigen-specific activation:

  • Transfer ovalbumin-specific CD4+ T cells into recipient mice and challenge with ovalbumin

  • Harvest spleens and use immunofluorescence to detect IMPDH2 filaments in transferred T cells

  • Co-stain with proliferation markers like Ki-67 to correlate filament formation with cell proliferation

• Inhibitor studies:

  • Treat activated T cells with IMPDH2 inhibitors like mycophenolic acid (MPA) or mycophenolate mofetil (MMF)

  • Use antibody pairs to detect changes in IMPDH2 localization and filament formation

  • Correlate with functional T-cell assays (e.g., cytokine production, proliferation)

Key findings:
Research has demonstrated abundant IMPDH2 filament formation during both in vitro and in vivo T-cell activation, establishing a correlation between IMPDH2 polymerization and lymphocyte activation . This model provides a valuable platform for investigating the molecular mechanisms and functional significance of IMPDH2 filament assembly in immune responses .

What are the current challenges in detecting IMPDH2 filaments in tissue samples, and how can they be overcome?

Detecting IMPDH2 filaments in tissue samples presents several technical challenges:

Challenges and solutions:

Advanced visualization strategies:

• Use live tissue imaging with IMPDH2-RFP fusion proteins to track dynamic filament formation

• Apply super-resolution microscopy techniques (STORM, PALM) for detailed filament structure

• Implement cleared tissue protocols (CLARITY, iDISCO) for 3D visualization of filaments in intact tissues

• Develop computational image analysis tools to quantify filament characteristics (length, thickness, branching)

Research in regenerating tadpole tails has demonstrated that IMPDH2 transiently localizes to cell membranes and punctae near amputation planes shortly after injury, with filamentous structures forming under specific conditions . This model provides insights for developing improved visualization techniques applicable to other tissue types.

What are common issues encountered when using IMPDH2 antibody pairs, and how can they be resolved?

Researchers frequently encounter several challenges when working with IMPDH2 antibody pairs:

Advanced troubleshooting approaches:

• Use antibody validation with IMPDH2 knockdown cells (via siRNA or shRNA) to confirm specificity

• Perform peptide competition assays to verify epitope specificity

• Compare results across different antibody clones/vendors to ensure consistency

• For filament studies, include known IMPDH2 filament-inducing conditions as positive controls

• Consider alternative detection methods (chemiluminescence vs. fluorescence) for optimizing signal-to-noise ratio

These strategies can significantly improve the reliability and reproducibility of experimental results when working with IMPDH2 antibody pairs.

How should researchers validate the specificity of IMPDH2 antibody pairs before experimental use?

Thorough validation of IMPDH2 antibody pairs is essential for reliable experimental results:

Comprehensive validation strategy:

• Western blot validation:

  • Test antibody against recombinant IMPDH2 protein

  • Compare signal in IMPDH2-expressing cell lines (e.g., HeLa, Jurkat, K562)

  • Perform knockdown validation using siRNA or shRNA against IMPDH2

  • Verify molecular weight (expected ~55.8 kDa)

• Cross-reactivity assessment:

  • Test against IMPDH1 (homologous isoform) to determine isoform specificity

  • Some antibodies may detect both IMPDH1 and IMPDH2

  • Verify species cross-reactivity if working with non-human samples

• Functional validation:

  • Confirm antibody pair works in intended applications (IP-WB, ELISA, etc.)

  • For IP-WB pairs, validate that the IP antibody effectively precipitates IMPDH2 and the WB antibody detects it

  • For ELISA pairs, confirm linearity of detection across a range of IMPDH2 concentrations

• Specificity controls:

  • Use IMPDH2 knockout/knockdown samples as negative controls

  • Include IMPDH2-overexpressing samples as positive controls

  • Perform peptide competition assays to confirm epitope specificity

A well-validated antibody pair should show consistent results across multiple experiments and detection methods, with signal intensity correlating with known IMPDH2 expression levels in different cell types or experimental conditions.

What quantitative methods can be used to analyze IMPDH2 filament formation and how should the data be interpreted?

Quantitative analysis of IMPDH2 filament formation requires systematic approaches:

Quantification methodologies:

• Immunofluorescence-based quantification:

  • Calculate percentage of cells containing IMPDH2 filaments

  • Measure filament length, thickness, and number per cell

  • Classify filaments as extended, bent, or compressed

  • Use intensity thresholding to identify filaments from background staining

• Image analysis parameters:

  • Measure mean fluorescence intensity of filaments versus diffuse IMPDH2

  • Quantify filament characteristics (length distribution, bundling, orientation)

  • Perform co-localization analysis with other cellular markers

  • Track dynamic changes in filament properties over time

• High-content screening approaches:

  • Automate filament detection across multiple samples/conditions

  • Develop classification algorithms for filament morphology

  • Correlate filament parameters with cellular phenotypes

Data interpretation framework:

• Baseline establishment: Determine normal range of filament occurrence in untreated cells (typically 0-10% in unstimulated T cells)

• Response indicators: Increased filament formation (40-60% in activated T cells) indicates cellular activation

• Dose-response relationships: Analyze how filament characteristics change with increasing inhibitor concentrations

• Temporal patterns: Examine changes in filament dynamics during cellular processes (regeneration, activation)

• Correlation with function: Link filament formation to enzyme activity measurements or cellular outcomes

Research has shown that IMPDH2 filament morphology correlates with enzyme activity states, with extended filaments representing active forms and compressed filaments indicating inhibited states . This structure-function relationship provides a framework for interpreting filament formation in various experimental contexts.

How can IMPDH2 antibody pairs be used to investigate the potential of IMPDH2 as a therapeutic target?

IMPDH2 is emerging as a promising therapeutic target, particularly in cancer and conditions involving excessive cell proliferation:

Target validation approaches:

• Expression correlation studies:

  • Use IMPDH2 antibody pairs in ELISA or IHC to quantify expression across disease states

  • Correlate expression levels with clinical outcomes (survival, treatment response)

  • Create tissue microarrays for high-throughput analysis of IMPDH2 expression in patient cohorts

• Inhibitor response monitoring:

  • Track changes in IMPDH2 expression, localization, and filament formation after treatment with inhibitors like MMF or MPA

  • Combine with functional assays (cell viability, apoptosis) to correlate molecular changes with cellular responses

  • Use IP-WB to investigate changes in IMPDH2 protein interactions following inhibitor treatment

• Synergistic therapy assessment:

  • Evaluate IMPDH2 inhibitors in combination with standard chemotherapeutics

  • Research has shown synergistic effects between MMF and doxorubicin (DOX)

  • Use antibody pairs to monitor how combined treatments affect IMPDH2 biology

Research findings and future directions:
Studies have demonstrated that knockdown of IMPDH2 significantly inhibits cell proliferation and induces cell cycle arrest at the G0/G1 phase . IMPDH2 overexpression correlates with adverse outcomes in certain cancers, and combining IMPDH2 inhibitors with conventional chemotherapy enhances therapeutic responses .

Future research should focus on developing more specific IMPDH2 inhibitors and identifying patient populations most likely to benefit from IMPDH2-targeted therapies based on expression profiles.

What is the significance of IMPDH2 filament formation in understanding guanine nucleotide regulation, and how can this be studied?

IMPDH2 filament formation represents a novel mechanism for regulating guanine nucleotide biosynthesis:

Regulatory significance:

• IMPDH2 filaments make the enzyme less sensitive to feedback inhibition by GTP

• This allows cells to maintain high guanine nucleotide synthesis during proliferative states

• Filament assembly is regulated by substrate (IMP) levels and can be modulated by proliferative signaling pathways like mTOR

Advanced study approaches:

• Structure-function analysis:

  • Use cryo-EM to determine high-resolution structures of IMPDH2 filaments in different states

  • Map conformational changes that occur during filament assembly

  • Identify key residues involved in filament formation through mutagenesis

• Allosteric regulation studies:

  • Compare enzyme kinetics between wildtype IMPDH2 and non-assembling mutants (e.g., Y12A)

  • Measure GTP inhibition sensitivity under various ATP concentrations

  • Analyze how substrate saturation affects filament stability and resistance to inhibition

• Metabolic integration:

  • Investigate how IMPDH2 filament formation responds to changes in cellular metabolism

  • Monitor filament dynamics during metabolic stress or nutrient deprivation

  • Link filament assembly to broader signaling networks controlling cell growth

Proposed model:
Research has established a model where IMPDH2 filament assembly modulates conformational changes to alter catalytic flux. Under physiologically high substrate (IMP) levels, assembled filaments resist the compressed, inhibited state, regardless of guanine levels . This creates a regulatory state where the enzyme can resist feedback inhibition during proliferative signaling , allowing for appropriate adjustment of guanine nucleotide levels according to metabolic demand.