AZI1 Antibody

What is AZI1 and what are its primary biological functions?

AZI1 (5-azacytidine induced 1), also called CEP131 or KIAA1118, is a multifunctional protein that plays important roles in several biological processes. In mammalian systems, AZI1 is located at the ciliary base and is required for cilium formation, potentially playing a role in spermatogenesis . In plant systems, AZI1 functions as a lipid transfer protein (LTP)-like molecule that is crucial for systemic immunity, particularly in the systemic movement of defense signals like azelaic acid (AZA) .

The protein contains distinct domains including an amino-terminal hydrophobic domain, a central proline-rich region (PRR) with unknown function, and a C-terminal LTP domain with eight cysteine motifs predicted to bind lipids . Due to this structure containing both a PRR and an 8CM domain, AZI1 is classified as a "Hybrid Proline Rich Protein" (HyPRP) .

What applications is the AZI1 antibody validated for?

The AZI1 antibody (25735-1-AP) has been validated for multiple experimental applications:

The antibody has been validated to show positive Western blot detection in mouse testis tissue and positive IF/ICC detection in HeLa cells . It shows reactivity with both human and mouse samples, making it suitable for comparative studies across these species .

What is the recommended protocol for Western blot using AZI1 antibody?

For optimal Western blot results using AZI1 antibody (25735-1-AP), researchers should follow these methodological steps:

• Prepare protein samples from appropriate tissues (mouse testis tissue shows positive results) or cell lines

• Resolve proteins using SDS-PAGE, considering that AZI1 has a calculated molecular weight of 122 kDa but observes at approximately 131 kDa

• Transfer proteins to a membrane using standard protocols

• Block the membrane with appropriate blocking buffer

• Dilute the primary AZI1 antibody in a range of 1:500 to 1:2000 depending on your sample type and detection system

• Incubate with appropriate secondary antibody and develop using your preferred detection system

• When analyzing results, note that the observed molecular weight is approximately 131 kDa, which is slightly higher than the calculated 122 kDa

For optimal results, it's recommended to validate dilution ratios for each specific experimental setup, as antibody performance can be sample-dependent .

How should AZI1 antibody be stored to maintain optimal activity?

The AZI1 antibody should be stored at -20°C where it remains stable for one year after shipment . The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is unnecessary for -20°C storage, which simplifies laboratory handling procedures . For the 20μl size variant, the preparation contains 0.1% BSA, which should be considered when planning experiments that might be sensitive to BSA presence .

Researchers should avoid repeated freeze-thaw cycles, which could potentially compromise antibody performance. When working with the antibody, it's advisable to keep it on ice and return it to -20°C storage promptly after use.

What controls should be included when using AZI1 antibody in immunofluorescence experiments?

When designing immunofluorescence experiments with AZI1 antibody, researchers should incorporate these essential controls:

• Negative controls:

  • Secondary antibody only (omitting primary AZI1 antibody) to assess background fluorescence

  • Isotype control (rabbit IgG) to detect non-specific binding

  • AZI1 knockdown/knockout cells or tissues (when available) to confirm specificity

• Positive controls:

  • HeLa cells, which have been validated to show positive IF/ICC detection with this antibody

  • Co-staining with established centrosome/ciliary base markers like gamma-tubulin, as demonstrated in validated immunostaining (AZI1 in red, gamma-tubulin in green)

• Subcellular localization validation:

  • Considering AZI1's known localization to ciliary bases, confirm appropriate subcellular distribution

  • In certain contexts, methanol fixation has been successfully used for AZI1 immunostaining with gamma-tubulin co-staining

The recommended dilution range for IF/ICC applications is 1:20-1:200, but this should be optimized for each specific experimental system to obtain optimal results .

How can researchers distinguish between different AZI1 subcellular populations?

AZI1 exhibits complex subcellular distribution patterns that vary between cell types and experimental models. To distinguish between different subcellular populations:

• In mammalian systems:

  • AZI1 primarily localizes to the ciliary base and functions in cilium formation

  • Co-staining with centrosomal markers (e.g., gamma-tubulin) can help distinguish this population

• In plant systems:

  • AZI1 shows dynamic localization to multiple compartments including the plastid envelope, endoplasmic reticulum (ER), plasma membrane (PM), and plasmodesmata

  • The proportion of AZI1 localized to plastids increases after systemic defense-triggering infections

  • Subcellular fractionation followed by Western blotting can be used to quantify distribution changes

  • Fluorescence microscopy with organelle-specific markers can visually distinguish different populations

• When using GFP-tagged constructs:

  • Various truncation or domain-specific constructs can help identify regions responsible for specific subcellular targeting

  • The N-terminal bipartite signature (TMD+PRR) is critical for plastid targeting in plants

  • Live-cell imaging can track trafficking between compartments

Understanding these distinct populations is essential for interpreting experimental results correctly, especially when studying AZI1's role in different cellular processes.

How do MPK3/6 kinases regulate AZI1 subcellular localization and function?

The mitogen-activated protein kinases MPK3 and MPK6 play crucial roles in modulating AZI1 subcellular localization, particularly in plant immune responses:

• Phosphorylation relationship:

  • MPK3/6 can phosphorylate AZI1 in vitro, suggesting direct post-translational modification

  • These kinases have prominent roles in systemic acquired resistance (SAR), induced systemic resistance (ISR), and defense priming induced by azelaic acid (AZA)

• Effect on AZI1 localization:

  • MPK3/6 promote the accumulation of AZI1 at plastids during priming induction

  • After pathogen or microbe-associated molecular pattern (MAMP) recognition, MPK3/6 are locally stimulated and enhance AZI1 plastid targeting through action on its N-terminal bipartite signal

  • This regulation appears to be specific to defense responses, suggesting targeted modulation during immunity activation

• Functional consequences:

  • The enhanced targeting to plastids likely determines the magnitude of systemic movement of defense signals for resistance and priming induction

  • This mechanism may ensure efficient movement of AZA for the establishment of systemic defenses

  • The regulation by MPK3/6 demonstrates how subcellular targeting of defense components can be dynamically controlled during immune responses

Researchers investigating AZI1 function should consider the activation status of these kinases, especially when comparing different treatment conditions or genotypes that might affect MPK3/6 activity.

What is the significance of AZI1's bipartite N-terminal targeting signal?

AZI1 utilizes a unique bipartite N-terminal targeting signal that represents a previously undescribed mechanism for protein localization to plastids:

This finding challenges conventional understanding of protein targeting mechanisms and has implications for predicting the localization of other proteins with similar structural features.

How can researchers investigate the relationship between AZI1 trafficking and microtubule networks?

To investigate the relationship between AZI1 trafficking and microtubule (MT) networks, researchers can employ several methodological approaches:

• Pharmacological interventions:

  • Treat cells with microtubule-disrupting agents (e.g., colchicine, nocodazole) or stabilizing agents (e.g., taxol)

  • Assess changes in AZI1 localization and trafficking dynamics using time-lapse microscopy with fluorescently-tagged AZI1

  • Compare trafficking rates and patterns before and after drug treatment

• Co-localization studies:

  • Perform dual-labeling experiments with AZI1 antibody and markers for microtubules (e.g., α-tubulin)

  • Analyze spatial and temporal relationships using super-resolution microscopy techniques

  • Quantify co-localization coefficients under different experimental conditions

• Live-cell imaging approaches:

  • Use GFP-tagged AZI1 constructs combined with fluorescently labeled tubulin to track movement along microtubules

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to measure trafficking kinetics

  • Analyze directionality and speed of AZI1-containing vesicles or complexes

• Genetic modifications:

  • Manipulate components of the microtubule cytoskeleton or motor proteins (e.g., kinesins, dyneins)

  • Assess the impact on AZI1 distribution using the AZI1 antibody in immunofluorescence applications

  • Compare wild-type with mutant cells to identify specific MT components required for AZI1 trafficking

These approaches can help determine whether AZI1 utilizes the MT network for intracellular trafficking as suggested in research , and identify the specific mechanisms involved in this process.

What could cause discrepancies between calculated and observed molecular weights of AZI1?

The AZI1 antibody detects the protein at approximately 131 kDa, which differs from the calculated molecular weight of 122 kDa . Several factors could explain this discrepancy:

• Post-translational modifications:

  • Phosphorylation: AZI1 can be phosphorylated by MPK3/6 kinases , which could add significant mass

  • Glycosylation or other modifications may alter the apparent molecular weight

  • The complex structure of AZI1 with multiple domains provides numerous potential modification sites

• Structural considerations:

  • The proline-rich region (PRR) in AZI1 may cause aberrant migration in SDS-PAGE

  • Proteins with high proline content often display abnormal migration patterns due to their rigid structure

  • The 8-cysteine motif (8CM) in the C-terminal LTP domain may form disulfide bonds that resist complete denaturation

• Technical factors:

  • Buffer conditions and reducing agent concentration can affect protein migration

  • Gel percentage and running conditions may influence the apparent molecular weight

  • Molecular weight standards used for calibration can introduce variability

To resolve whether the discrepancy represents actual biological variation or technical artifacts, researchers could:

• Compare migration patterns under different reducing conditions

• Analyze AZI1 from different tissues or after treatment with phosphatases

• Use mass spectrometry to determine the exact mass and identify specific modifications

What are common sources of non-specific binding with AZI1 antibody and how can they be minimized?

When working with AZI1 antibody, researchers may encounter non-specific binding that complicates data interpretation. Common sources and mitigation strategies include:

• Antibody concentration issues:

  • Using too high a concentration of primary antibody can increase background

  • Titrate the antibody within the recommended ranges (WB: 1:500-1:2000, IF/ICC: 1:20-1:200)

  • Perform pilot experiments to determine optimal concentration for each specific sample type

• Blocking optimization:

  • Insufficient blocking can lead to non-specific binding

  • Optimize blocking buffer composition (BSA vs. milk vs. commercial alternatives)

  • Extend blocking time for challenging samples

• Cross-reactivity concerns:

  • The polyclonal nature of the AZI1 antibody (25735-1-AP) may result in recognition of epitopes shared with other proteins

  • Include appropriate negative controls such as AZI1 knockdown/knockout samples

  • For critical experiments, validate findings with a second antibody targeting a different epitope

• Fixation and sample preparation effects:

  • Different fixation methods can affect epitope accessibility and non-specific binding

  • For IF/ICC applications, compare methanol fixation (which has been validated) with alternatives

  • Ensure complete permeabilization while minimizing structural disruption

• Detection system optimization:

  • Secondary antibody dilution should be optimized alongside primary antibody

  • Consider using highly cross-adsorbed secondary antibodies to reduce species cross-reactivity

  • For low-abundance targets, balance signal amplification against increased background

Thorough documentation of optimization steps will help establish reliable protocols for consistent results across experiments.

How can researchers validate AZI1 antibody specificity in their experimental system?

Validating antibody specificity is critical for generating reliable scientific data. For AZI1 antibody, researchers should consider these methodological approaches:

• Genetic validation approaches:

  • Utilize AZI1 knockdown (siRNA/shRNA) or knockout (CRISPR/Cas9) systems

  • Compare signal intensity in Western blot or immunofluorescence between control and knockdown/knockout samples

  • The AZI1 antibody has been validated in knockdown/knockout systems in 3 published studies

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide/antigen (AZI1 fusion protein Ag22699)

  • Compare signal with and without peptide competition

  • Specific signals should be diminished or eliminated after peptide competition

• Multiple detection methods:

  • Correlate protein detection by Western blot with localization by immunofluorescence

  • Verify that subcellular localization matches expected patterns (e.g., ciliary base in mammalian cells )

  • Co-staining with established markers (e.g., gamma-tubulin for centrosomes) can provide additional validation

• Heterologous expression systems:

  • Overexpress tagged versions of AZI1 and confirm detection with both the tag antibody and AZI1 antibody

  • Test antibody in cells normally negative for AZI1 expression with and without transfection

  • Compare detection patterns between endogenous and overexpressed protein

• Cross-species validation:

  • The antibody shows reactivity with both human and mouse samples

  • Verify specificity in the particular species being studied

  • Consider evolutionary conservation when interpreting cross-species reactivity

These validation steps should be performed in the specific experimental system being used to ensure reliable interpretation of results.

How might AZI1 function differ between plant and mammalian systems?

AZI1 appears to have evolved specialized functions across different biological systems while maintaining some core mechanistic features:

• Functional divergence:

  • In mammalian systems, AZI1 (also called CEP131) is primarily involved in ciliogenesis, localizing to the ciliary base and contributing to cilium formation

  • In plant systems, AZI1 functions as a key component of systemic immunity, particularly in the movement of defense signals like azelaic acid (AZA)

  • These distinct functions reflect adaptation to the specific needs of each biological system

• Localization similarities and differences:

  • Mammalian AZI1 localizes primarily to centrosomes/ciliary bases

  • Plant AZI1 shows more complex localization to multiple compartments including plastid envelopes, endoplasmic reticulum, plasma membrane, and plasmodesmata

  • Both systems show dynamic regulation of AZI1 localization in response to stimuli

• Molecular mechanisms:

  • Both systems likely involve regulation by phosphorylation: mammalian AZI1 is phosphorylated during cell cycle progression, while plant AZI1 is phosphorylated by defense-associated MPK3/6 kinases

  • The bipartite targeting mechanism discovered in plants may have parallels in mammalian cells, though this remains to be fully investigated

  • The lipid-binding capabilities of the C-terminal domain may be functionally relevant in both systems

Researchers should be cautious when extrapolating findings between plant and mammalian systems, while also recognizing potential opportunities for cross-system insights.

What methodological approaches can determine if AZI1 forms functional complexes with other proteins?

To investigate AZI1's protein interaction partners and complex formation, researchers can employ these complementary methodological approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use the AZI1 antibody for immunoprecipitation, which has been validated in 2 publications

  • Perform reverse Co-IP with antibodies against suspected interaction partners

  • Analyze complexes by Western blot or mass spectrometry

  • Compare interactions under different physiological conditions (e.g., control vs. pathogen infection in plants)

• Proximity-based labeling approaches:

  • Generate BioID or TurboID fusions with AZI1 to identify proximal proteins

  • APEX2 fusion constructs can provide temporal resolution of interaction dynamics

  • These approaches are particularly valuable for membrane-associated proteins like AZI1

• Fluorescence-based interaction assays:

  • Förster Resonance Energy Transfer (FRET) between AZI1 and potential partners

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in situ

  • Fluorescence Correlation Spectroscopy (FCS) to analyze complex formation kinetics

• Crosslinking mass spectrometry:

  • Chemical crosslinking followed by mass spectrometry can capture transient interactions

  • Particularly useful for membrane-associated complexes that may be disrupted by detergents

  • Can provide structural insights into the arrangement of proteins within complexes

• Functional validation of interactions:

  • Mutational analysis of interaction interfaces

  • Phenotypic rescue experiments with interaction-deficient mutants

  • Correlation of complex formation with functional outcomes (e.g., systemic immunity in plants or cilia formation in mammalian cells)

These approaches can reveal how AZI1 functions within larger protein complexes to mediate its diverse biological roles.

How can researchers investigate the role of AZI1 in cross-membrane transport of lipid signals?

AZI1's predicted lipid-binding capacity through its C-terminal LTP domain and its localization to membrane interfaces suggest potential roles in lipid transport. To investigate this function, researchers could employ these methodological approaches:

• Lipid binding assays:

  • In vitro lipid binding assays with purified AZI1 protein or specific domains

  • Lipid overlay assays to determine binding specificity

  • Surface plasmon resonance to measure binding kinetics for different lipid species

  • Focus particularly on azelaic acid (AZA) binding in plant systems

• Transport assays:

  • Generate liposomes with different lipid compositions to model membrane interfaces

  • Measure transfer of fluorescently-labeled lipids between membrane compartments

  • Compare wild-type AZI1 with mutants in the lipid-binding domain

  • Correlate transport activity with biological function (e.g., systemic immunity)

• Imaging approaches:

  • Visualize AZI1 at membrane contact sites between plastids and ER in plants

  • Track fluorescently-labeled lipids in relation to AZI1 localization

  • Employ super-resolution microscopy to resolve detailed membrane interactions

  • Perform FRAP experiments to measure dynamics at contact sites

• Genetic manipulation strategies:

  • Generate targeted mutations in the LTP domain to disrupt lipid binding

  • Assess the impact on AZI1 localization and function

  • Evaluate the ability of mutant constructs to complement azi1 knockout phenotypes

  • Create chimeric proteins to test domain-specific functions

These approaches can help elucidate whether AZI1 directly participates in non-vesicular transport of AZA and possibly other non-polar signals to systemic tissues as proposed in plant immunity research .

How might understanding AZI1 function contribute to broader research fields?

Research on AZI1 has potential implications across multiple scientific disciplines:

• Cellular biology:

  • AZI1 provides a model for studying unconventional protein targeting mechanisms

  • Its dynamic localization offers insights into how cells regulate protein trafficking in response to stimuli

  • The bipartite targeting signal represents a novel class of signal-anchored proteins

• Plant immunity:

  • AZI1's role in systemic acquired resistance enhances understanding of long-distance signaling in plants

  • Its regulation by defense-associated kinases MPK3/6 illuminates how immune responses are coordinated

  • The connection to azelaic acid transport links lipid signaling to systemic immunity

• Mammalian cell biology:

  • AZI1's function at ciliary bases contributes to understanding ciliogenesis mechanisms

  • Its potential role in spermatogenesis connects to reproductive biology research

  • Studying AZI1 regulation may provide insights into ciliopathies and related disorders

Understanding these diverse functions could lead to applications in agriculture (enhancing plant disease resistance) and medicine (addressing ciliopathy-related conditions), highlighting the value of basic research on this multifunctional protein.