At1g68110 Antibody

What is At1g68110 and why is it significant for plant research?

At1g68110 encodes a clathrin assembly protein in Arabidopsis thaliana that has been identified to possess adenylyl cyclase (AC) activity. This dual functionality makes it particularly interesting for researchers studying plant signaling pathways. The protein generates cAMP and shows substrate preference for ATP rather than GTP, with Mn²⁺-dependent activity that is significantly enhanced by Ca²⁺ . The presence of both clathrin assembly and adenylyl cyclase functions in a single protein suggests potential roles in coordinating membrane trafficking events with cellular signaling cascades, representing an important area of investigation in plant cell biology.

What applications is the At1g68110 antibody validated for?

The At1g68110 antibody has been specifically validated for ELISA and Western blot (WB) applications to ensure proper identification of the antigen . These techniques allow researchers to detect and quantify At1g68110 protein expression in plant tissue samples. For Western blot applications, the antibody recognizes the native protein under reducing conditions, making it suitable for detecting protein expression patterns across different plant tissues, developmental stages, or in response to various experimental treatments. The antibody has not been validated for immunohistochemistry or immunofluorescence applications, so researchers interested in these techniques would need to perform additional validation studies.

What are the optimal storage and handling conditions for At1g68110 antibody?

For maximum stability and activity retention, the At1g68110 antibody should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can compromise antibody functionality. The antibody is supplied in liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . This formulation enhances stability during storage. For working solutions, aliquoting the antibody into single-use volumes is recommended to prevent protein degradation from multiple freeze-thaw cycles. Typical working dilutions will depend on the specific application and should be determined empirically, though manufacturer's recommendations usually provide a starting range.

How is the specificity of At1g68110 antibody confirmed?

The specificity of At1g68110 antibody is confirmed through multiple validation steps. As an antigen-affinity purified polyclonal antibody, it undergoes rigorous testing to ensure it specifically recognizes the target protein . Validation typically includes:

• Western blot analysis - Demonstrating a single band of appropriate molecular weight in Arabidopsis thaliana extracts

• ELISA testing - Showing specific binding to recombinant At1g68110 protein

• Negative controls - Testing against samples known not to express the target protein

• Cross-reactivity assessment - Evaluating potential binding to related proteins

Researchers should perform additional validation when using the antibody in new experimental contexts or with modified protocols.

How can At1g68110 antibody be used to investigate the dual functionality of AtClAP in plant signaling?

The At1g68110 antibody serves as a critical tool for investigating the dual functionality of AtClAP in plant signaling through several sophisticated experimental approaches:

• Co-immunoprecipitation (Co-IP) studies: By using the At1g68110 antibody for immunoprecipitation followed by mass spectrometry, researchers can identify protein interaction partners that associate with AtClAP during clathrin-mediated endocytosis or cAMP signaling events. This approach can reveal how AtClAP transitions between its distinct functional roles.

• Subcellular fractionation coupled with immunoblotting: Researchers can fractionate plant cells into membrane, cytosolic, and nuclear components, then use the At1g68110 antibody for Western blot analysis to determine the protein's localization under different conditions, revealing when and where each function predominates.

• Proximity labeling assays: By combining the At1g68110 antibody with proximity labeling techniques such as BioID or APEX, researchers can map the protein's dynamic interaction network in living cells during specific cellular processes.

• Phosphorylation state analysis: The antibody can be used to immunoprecipitate AtClAP for subsequent phosphoproteomic analysis to determine how post-translational modifications regulate the switch between clathrin assembly and adenylyl cyclase activities.

The relationship between Ca²⁺ modulation of adenylyl cyclase activity and membrane trafficking events presents a particularly promising research direction, as the data indicates AtClAP's enzymatic function is significantly enhanced by Ca²⁺ .

What experimental controls should be included when using At1g68110 antibody for quantitative analysis?

When using At1g68110 antibody for quantitative analysis, several critical controls must be included to ensure reliable and reproducible results:

• Negative controls:

  • Samples from Arabidopsis knockout mutants lacking the At1g68110 gene

  • Secondary antibody-only controls to assess background signal

  • Pre-immune serum controls to evaluate non-specific binding

• Positive controls:

  • Recombinant At1g68110 protein at known concentrations for standard curve generation

  • Samples from plants overexpressing At1g68110 under a constitutive promoter

• Loading controls:

  • For Western blot analysis, probing for housekeeping proteins like actin or tubulin

  • For immunoprecipitation studies, analysis of input, unbound, and eluate fractions

• Signal validation controls:

  • Peptide competition assays where the antibody is pre-incubated with excess immunizing peptide

  • Dilution series to confirm signal linearity within the working range

• Technical replicates:

  • At least three independent experiments with multiple technical replicates per condition

  • Statistical analysis to determine significance of observed differences

These controls are essential for disambiguating specific signal from background noise and for accurate quantification of At1g68110 protein levels across different experimental conditions.

How can researchers optimize immunoprecipitation protocols for At1g68110 protein complexes?

Optimizing immunoprecipitation (IP) protocols for At1g68110 protein complexes requires careful consideration of multiple parameters to preserve native protein interactions while achieving high specificity and yield:

• Lysis buffer optimization:

  • Test multiple buffer compositions varying in ionic strength (150-300 mM NaCl)

  • Evaluate different detergents (0.1-1% NP-40, Triton X-100, or digitonin)

  • Include appropriate protease and phosphatase inhibitors

  • Assess the effect of calcium chelators or calcium supplementation given the Ca²⁺-dependency of AtClAP activity

• Antibody coupling strategies:

  • Direct coupling to magnetic or agarose beads using crosslinkers

  • Pre-incubation of antibody with lysate followed by Protein A/G capture

  • Comparison of different antibody:lysate ratios (1:10 to 1:100)

• Incubation conditions:

  • Test various temperatures (4°C, room temperature)

  • Optimize incubation time (1 hour to overnight)

  • Compare static vs. rotational mixing

• Washing stringency:

  • Develop a progressive washing strategy with increasing salt concentrations

  • Determine the minimum number of washes required to reduce background

• Elution methods:

  • Compare competitive elution with immunizing peptide

  • Evaluate pH-based elution (glycine buffer pH 2.5-3.0)

  • Test denaturing elution with SDS sample buffer

A sample optimization matrix is presented below:

The optimal protocol will depend on the specific downstream application and whether the focus is on capturing the clathrin assembly function or the adenylyl cyclase activity of the protein.

What approaches can be used to investigate the relationship between At1g68110's clathrin assembly and adenylyl cyclase functions?

Investigating the relationship between At1g68110's dual functions requires sophisticated experimental approaches that can temporally and spatially resolve these activities:

• Domain-specific functional assays:

  • Generate recombinant proteins containing either the clathrin assembly domain or the adenylyl cyclase domain

  • Compare activity profiles using in vitro reconstitution assays

  • Develop domain-specific antibodies to track each function independently

• Real-time imaging with biosensors:

  • Use FRET-based cAMP biosensors in Arabidopsis to monitor adenylyl cyclase activity

  • Simultaneously track clathrin-coated vesicle formation with fluorescently-tagged clathrin

  • Correlate temporal relationships between clathrin assembly and cAMP production

• Conditional regulation experiments:

  • Design experimental systems where calcium levels can be manipulated, as AtClAP activity is Ca²⁺-enhanced

  • Monitor both clathrin assembly and cAMP production under varying calcium concentrations

  • Use calcium chelators or ionophores to determine whether calcium modulation affects both functions equally

• Structure-function analysis:

  • Perform site-directed mutagenesis to identify key residues involved in each function

  • Express mutant versions in at1g68110 knockout plants

  • Use the At1g68110 antibody to confirm expression levels

  • Assess how mutations affecting one function impact the other

• Induced proximity experiments:

  • Develop systems to artificially induce AtClAP dimerization or membrane recruitment

  • Determine whether induced changes in localization or protein conformation differentially affect its dual functions

The relationship between adenylyl cyclase activity and clathrin assembly function might represent a novel mechanism for coordinating membrane trafficking with cellular signaling in plants, potentially regulated by calcium as a common modulator of both activities.

How can At1g68110 antibody be used to study the protein's role in stress response pathways?

At1g68110 antibody provides a valuable tool for investigating this protein's role in plant stress response pathways through several experimental approaches:

• Stress-induced expression profiling:

  • Subject Arabidopsis plants to various stresses (drought, salt, pathogens, temperature)

  • Harvest tissue at multiple time points post-stress

  • Use the At1g68110 antibody for Western blot analysis to quantify changes in protein expression

  • Correlate protein levels with physiological and molecular stress markers

• Post-translational modification analysis:

  • Immunoprecipitate At1g68110 from stressed and unstressed plants

  • Analyze samples using phospho-specific staining or mass spectrometry

  • Identify stress-specific modifications that might regulate protein function

  • Generate a temporal map of modifications in response to stress progression

• Protein-protein interaction studies under stress conditions:

  • Perform co-immunoprecipitation using At1g68110 antibody under normal and stress conditions

  • Identify differential interaction partners using mass spectrometry

  • Validate key interactions using reciprocal co-IP or proximity labeling techniques

  • Map interaction networks specific to different stress conditions

• Subcellular relocalization tracking:

  • Combine cell fractionation with At1g68110 immunoblotting

  • Determine if stress induces changes in the protein's subcellular distribution

  • Correlate relocalization events with changes in clathrin assembly or adenylyl cyclase activity

• Correlation with cAMP signaling dynamics:

  • Measure cellular cAMP levels in response to stress

  • Use the At1g68110 antibody to track protein abundance and activity state

  • Determine whether stress-induced changes in cAMP correlate with At1g68110 regulation

The data from these experiments would provide insights into whether At1g68110 functions as a stress response coordinator by linking membrane trafficking (through its clathrin assembly function) with cAMP signaling (through its adenylyl cyclase activity).

What are the key differences between polyclonal and monoclonal antibodies for At1g68110 research?

The At1g68110 antibody available from commercial sources is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At1g68110 protein . Understanding the implications of using polyclonal versus theoretical monoclonal antibodies for this target is important for experimental design:

Polyclonal At1g68110 antibody characteristics:

• Recognizes multiple epitopes on the At1g68110 protein

• Provides robust signal due to binding of multiple antibodies per target molecule

• Offers greater tolerance to minor protein denaturation or conformational changes

• May exhibit batch-to-batch variation requiring validation between lots

• Currently available as antigen-affinity purified preparations to reduce non-specific binding

Theoretical monoclonal At1g68110 antibody considerations:

• Would recognize a single epitope with high specificity

• Would provide consistent performance between batches with minimal variation

• Might be less tolerant to protein modifications or conformational changes

• Could potentially miss detecting certain isoforms or post-translationally modified versions

• Would require extensive screening to identify clones recognizing functionally relevant epitopes

How does the adenylyl cyclase activity of At1g68110 compare to other plant adenylyl cyclases?

The adenylyl cyclase (AC) activity of At1g68110 displays several distinctive characteristics when compared to other plant adenylyl cyclases:

• Catalytic efficiency: Recombinant At1g68110 AC domain (amino acids 261-379) generates approximately 110 fmols/μg protein of cAMP in 15 minutes under optimal conditions with Mn²⁺, and about 31 fmols/μg protein with Mg²⁺ . This activity level is within the range typical for plant ACs but lower than animal ACs.

• Cofactor preference: At1g68110 shows strong preference for Mn²⁺ over Mg²⁺ as a cofactor, with approximately 3.5-fold higher activity observed with Mn²⁺ . This metal ion preference is common among plant adenylyl cyclases.

• Calcium modulation: The AC activity of At1g68110 is significantly enhanced by Ca²⁺ , a feature shared with some other plant enzymes such as the Phytosulfokine receptor (AtPSKR1), where Ca²⁺ acts as a molecular switch between kinase and guanylyl cyclase activities.

• Substrate specificity: At1g68110 demonstrates preference for ATP over GTP as substrate , confirming its classification as an adenylyl rather than guanylyl cyclase.

• Structural context: Unlike canonical animal adenylyl cyclases, At1g68110 integrates AC activity within a protein primarily characterized as a clathrin assembly protein, representing a novel functional combination.

The comparative enzymatic parameters of At1g68110 and other plant adenylyl cyclases are summarized in the table below:

This comparison highlights At1g68110's relatively high specific activity among plant adenylyl cyclases and its distinctive regulation by calcium, suggesting specialized roles in calcium-dependent signaling pathways potentially linked to membrane trafficking events.

How might advancements in antibody engineering impact future research on At1g68110?

Recent advancements in antibody engineering technologies could significantly enhance future research on At1g68110 through several innovative approaches:

• Development of fragment antibodies: Smaller antibody formats such as single-chain variable fragments (scFvs) or nanobodies derived from the At1g68110 antibody could provide superior tissue penetration for in vivo imaging studies . These engineered formats maintain specificity while enabling access to subcellular compartments that might be inaccessible to conventional antibodies.

• Domain-specific antibodies: Engineering antibodies that specifically recognize either the clathrin assembly domain or the adenylyl cyclase domain of At1g68110 would allow researchers to distinguish between these functional aspects in situ. This approach could reveal the spatial and temporal regulation of each activity within living plant cells.

• Conformation-specific antibodies: Advanced antibody engineering could produce variants that selectively bind to At1g68110 in specific conformational states, potentially distinguishing between active and inactive forms of its adenylyl cyclase function. Such tools would be valuable for understanding the protein's activation mechanisms.

• Bispecific antibodies: Engineered antibodies that simultaneously bind At1g68110 and one of its interaction partners could be developed to study specific protein complexes. This approach would enable selective analysis of subpopulations of the protein engaged in particular cellular functions.

• Antibody-based biosensors: Integration of the At1g68110 antibody binding domain with fluorescent reporters could generate biosensors that report on protein conformation, post-translational modifications, or interaction states in real-time within living plants.

Modern protein engineering platforms, including AI-assisted antibody design , could accelerate the development of these specialized research tools, potentially revealing new insights into how At1g68110 coordinates membrane trafficking with cellular signaling pathways.

What is the significance of studying At1g68110 in the context of plant stress responses and climate adaptation?

Investigating At1g68110's role in plant stress responses could yield significant insights relevant to climate adaptation strategies:

• Signaling hub integration: As a protein with dual functionality in clathrin-mediated endocytosis and cAMP production, At1g68110 potentially serves as an integration point between membrane receptor dynamics and downstream signaling. This position would be strategic for coordinating responses to environmental stresses.

• Drought and osmotic stress adaptation: Clathrin-mediated endocytosis regulates the abundance of membrane transporters and channels involved in water and ion homeostasis. At1g68110's role in this process, coupled with its ability to generate the secondary messenger cAMP, may coordinate adaptive responses to water deficit conditions.

• Pathogen response regulation: Plant immune receptors undergo endocytosis following pathogen perception, a process requiring clathrin assembly proteins. At1g68110's potential involvement in this process, combined with its adenylyl cyclase activity, might link receptor internalization with defense signaling activation.

• Temperature stress signaling: Calcium signaling is critical in temperature stress responses, and At1g68110's calcium-enhanced adenylyl cyclase activity suggests it may function in calcium-cAMP signaling cascades during temperature extremes.

• Cross-talk with hormone pathways: cAMP has been implicated in modulating hormone responses in plants. At1g68110's adenylyl cyclase activity may influence hormone-mediated stress adaptation through cAMP production.

Understanding these mechanisms could inform strategies for engineering climate-resilient crops by targeting specific signaling nodes that coordinate multiple adaptation pathways. This research direction becomes increasingly relevant as climate change intensifies environmental stresses on agricultural systems worldwide.