AHP5 Antibody

What is AHP5 and why are antibodies against it important for plant research?

AHP5 is one of the five histidine phosphotransfer proteins in Arabidopsis thaliana that function in multi-step phosphorelay signal transduction systems. These proteins contain a highly conserved XHQXKGSSXS motif with a histidine phosphorylation site and act as intermediaries in the two-component signaling pathway . Anti-AHP5 antibodies are crucial research tools for studying plant internal clock and multistep phosphorelay signaling in Arabidopsis thaliana. They enable researchers to track protein expression, localization, and interactions, providing insights into signaling mechanisms that regulate plant growth and stress responses .

How does AHP5 differ functionally from other Arabidopsis histidine phosphotransfer proteins?

While all five AHPs (AHP1-AHP5) in Arabidopsis share similar structures and generally function as redundant positive regulators in cytokinin signaling, they display distinct roles in specific pathways. Research has shown that AHP2, AHP3, and AHP5 have redundant but negative regulatory roles in drought stress response, with evidence suggesting that AHP2 and AHP3 might play more prominent roles than AHP5 . The expression patterns of these genes under stress conditions also differ significantly - AHP5 uniquely shows a slight increase at 1 hour after abscisic acid treatment before rapidly decreasing, a pattern distinct from AHP2 and AHP3 .

What is the current understanding of AHP5 subcellular localization?

Contrary to earlier models suggesting that AHPs relocalize to the nucleus in response to cytokinin, research has demonstrated that AHP proteins, including AHP5, actively maintain a consistent nuclear/cytosolic distribution regardless of cytokinin signaling status . The study showed that AHPs maintain this distribution by balancing active transport into and out of the nucleus. This finding challenges previous assumptions and has important implications for understanding how cytokinin signaling is transmitted from receptors to nuclear response regulators .

What specific research applications are AHP5 antibodies suitable for?

According to published data, AHP5 antibodies are versatile tools for multiple experimental applications:

• Western blotting (recommended dilution 1:10,000)

• Enzyme-linked immunosorbent assay (ELISA)

• Protein array analysis

• Investigation of protein-protein interactions

• Pull-down assays

• Affinity purification

• Subcellular localization studies

• Detection of splicing variants

These antibodies are suitable for both qualitative investigation of magnesium-dependent phosphotransfer relay and quantification of particular sensor histidine kinases .

How can AHP5 antibodies be used to study plant stress response mechanisms?

AHP5, along with AHP2 and AHP3, has been implicated in drought stress responses in Arabidopsis. Research has shown that the expression of AHP5 is down-regulated by dehydration, high salinity, and abscisic acid treatment . The ahp2,3,5 triple mutant exhibits a strong drought-tolerant phenotype associated with stimulation of protective mechanisms . AHP5 antibodies can be used to:

• Track changes in AHP5 protein levels during stress responses

• Compare protein expression with transcript levels to identify post-transcriptional regulation

• Examine protein modifications that may occur under stress conditions

• Identify interaction partners that may change during stress adaptation

• Investigate subcellular redistribution in response to stress signals

Additionally, AHP5 is involved in stomatal closure in response to hydrogen peroxide and ethylene as part of a complex TCS signaling network . AHP5 antibodies can help elucidate these signaling pathways in guard cells and understand how plants regulate water loss through stomata under different conditions.

How can AHP5 antibodies be applied in protein-protein interaction studies?

AHP5 interacts with various proteins in the two-component signaling pathway, including histidine kinases like AHK5 and response regulators. AHP5 antibodies can be employed for:

• Co-immunoprecipitation to identify novel interaction partners

• Pull-down assays to confirm direct protein-protein interactions

• Protein array analysis to screen for potential interactors

• In situ proximity ligation assays to visualize interactions in plant tissues

• Chromatin immunoprecipitation if AHP5 is found to associate with DNA-binding proteins

Research has demonstrated that AHP5 interacts with the histidine kinase CKI1 receiver domain, although this interaction appears to be relatively weak compared to other AHP-CKI1 interactions . This finding illustrates how antibodies can help characterize the specificity and strength of protein-protein interactions in signaling networks.

What are the optimal storage conditions for maintaining AHP5 antibody activity?

For maximum stability and activity retention, AHP5 antibodies should be:

• Flash frozen in liquid nitrogen immediately after purification

• Stored at -80°C for long-term preservation

• Kept in appropriate buffer conditions (50 mM Tris-HCl pH 8 with 20% glycerol at 1 mg/ml concentration)

• Protected from repeated freeze-thaw cycles, which significantly reduce antibody effectiveness

Aliquoting the antibody upon receipt into single-use volumes can help prevent degradation from multiple freeze-thaw cycles and maintain consistency across experiments.

What controls should be incorporated in experimental designs using AHP5 antibodies?

To ensure reliable and interpretable results when using AHP5 antibodies, researchers should implement multiple controls:

• Positive controls: Wild-type Arabidopsis tissues expressing AHP5 or recombinant AHP5 protein

• Negative controls:

  • Tissues from ahp5 knockout mutants

  • Samples where primary antibody is omitted

  • Pre-immune serum controls

• Specificity controls:

  • Competition assays with purified AHP5 protein

  • Testing for cross-reactivity with other AHP proteins (particularly important given the sequence similarity among AHP1-5)

• Loading controls: Use of housekeeping proteins for normalization in Western blot analysis

• Antibody validation: Use of different antibodies targeting different epitopes of AHP5 to confirm results

These controls help distinguish specific from non-specific binding and validate experimental findings.

How should researchers approach quantitative analysis of AHP5 protein expression?

For accurate quantitative analysis of AHP5 protein levels, researchers should consider:

• Western blotting quantification:

  • Use appropriate loading controls (e.g., actin, tubulin)

  • Include standard curves with known amounts of recombinant AHP5

  • Use digital imaging systems with linear dynamic range

  • Perform biological and technical replicates (minimum n=3)

• ELISA-based quantification:

  • Develop standard curves with purified AHP5 protein

  • Optimize antibody concentrations to ensure operation within the linear range

  • Use consistent protein extraction protocols across all samples

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Consider normality of data distribution

  • Use paired tests when comparing treatments on the same plant material

• Normalization strategies:

  • Normalize to total protein content

  • Consider cell-type specific markers when working with complex tissues

  • Account for developmental stage differences that might affect baseline expression

What are common challenges when using AHP5 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with AHP5 antibodies:

• Cross-reactivity with other AHP proteins:

  • Solution: Perform specificity tests using recombinant AHP proteins

  • Use peptide competition assays with specific AHP5 peptides

  • Validate with genetic controls (e.g., ahp5 mutants)

• Low signal strength:

  • Solution: Optimize antibody concentration and incubation conditions

  • Use signal enhancement systems compatible with your detection method

  • Increase protein loading if appropriate

  • Consider protein enrichment through immunoprecipitation before detection

• High background:

  • Solution: Increase blocking time and concentration

  • Optimize washing steps (number, duration, buffer composition)

  • Try different blocking agents (BSA, milk, commercial alternatives)

  • Dilute antibody further if concentration is too high

• Inconsistent results between experiments:

  • Solution: Standardize plant growth conditions

  • Use consistent tissue harvesting and protein extraction protocols

  • Prepare larger batches of working antibody dilutions to use across experiments

  • Include internal reference samples across all experiments

How should researchers interpret apparently contradictory results between AHP5 transcript and protein levels?

Discrepancies between AHP5 mRNA expression and protein levels are not uncommon and can provide valuable insights into regulatory mechanisms:

• Post-transcriptional regulation: MicroRNAs or RNA-binding proteins may affect translation efficiency of AHP5 mRNA without changing transcript levels

• Protein stability differences: Environmental conditions may affect the half-life of AHP5 protein without altering transcription rates. For example, stress conditions might trigger post-translational modifications that stabilize or destabilize the protein

• Temporal dynamics: Consider that there is typically a time lag between changes in transcription and corresponding changes in protein levels. Time-course experiments can help resolve apparent contradictions

• Tissue-specific effects: Whole-tissue analysis may mask cell type-specific changes. Consider using techniques like immunohistochemistry to examine spatial distribution

• Technical considerations: Different sensitivities of transcript detection (e.g., qPCR) versus protein detection (e.g., Western blot) methods can create apparent discrepancies

In one study, researchers found that AHP5 transcripts decreased under stress conditions , but protein-level analysis might reveal different dynamics due to post-translational regulation mechanisms.

How can researchers distinguish between specific effects on AHP5 versus general effects on the cytokinin signaling pathway?

Distinguishing AHP5-specific effects from general cytokinin pathway effects requires careful experimental design:

• Genetic approach: Compare phenotypes and molecular responses across single and higher-order ahp mutants:

  • ahp5 single mutant

  • ahp2,5 and ahp3,5 double mutants

  • ahp2,3,5 triple mutant

  • Other higher-order combinations

• Biochemical approach:

  • Use AHP5 antibodies to immunoprecipitate specific protein complexes

  • Compare with complexes isolated using antibodies against other AHPs

  • Identify unique versus shared interaction partners

• Pharmacological approach:

  • Apply cytokinin receptor inhibitors or agonists

  • Monitor AHP5-specific responses versus general pathway responses

  • Use phosphorylation-specific antibodies if available

• Tissue-specific analysis:

  • Investigate whether effects are more pronounced in tissues where AHP5 expression is highest

  • Compare with expression patterns of other AHPs

Research has shown that while AHP5 functions redundantly with other AHPs in many contexts, it also displays unique expression patterns under stress conditions that distinguish it from AHP2 and AHP3 .

How can AHP5 antibodies be used to study cross-talk between different signaling pathways?

AHP5 functions at the intersection of multiple signaling pathways, making antibodies against it valuable for studying signaling cross-talk:

• Cytokinin and drought stress pathway interactions:

  • AHP5, along with AHP2 and AHP3, functions as a negative regulator of drought responses

  • AHP5 antibodies can help track protein abundance and modifications during combined hormonal and stress treatments

  • Co-immunoprecipitation with AHP5 antibodies can identify stress-specific interaction partners

• Hydrogen peroxide and ethylene signaling integration:

  • AHP5 has been identified as acting downstream of AHK5 in ethylene and hydrogen peroxide response pathways in guard cells

  • Antibodies can help investigate the molecular mechanisms by which these pathways converge

• Abscisic acid (ABA) and cytokinin antagonism:

  • AHP5 transcript levels respond to ABA treatment

  • Protein-level analysis using antibodies can reveal post-translational regulatory mechanisms

  • Immunoprecipitation followed by mass spectrometry can identify ABA-dependent modifications or interactions

• Two-component and MAPK pathway connections:

  • Investigate whether AHP5 interacts with components of MAPK cascades under specific conditions

  • Use antibodies to track co-localization or complex formation between signaling components

What approaches can be used to study AHP5 phosphorylation dynamics in planta?

Understanding AHP5 phosphorylation dynamics is crucial for deciphering its role in signal transduction:

• Phosphorylation-specific techniques:

  • Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated AHP5

  • Immunoprecipitation with AHP5 antibodies followed by phospho-specific staining

  • Mass spectrometry analysis of immunoprecipitated AHP5 to identify phosphorylation sites

• Time-course experiments:

  • Track AHP5 phosphorylation status at different times after stimulation

  • Compare phosphorylation dynamics across different tissues or developmental stages

  • Correlate phosphorylation changes with downstream responses

• Genetic approaches:

  • Express phospho-mimetic or phospho-null AHP5 variants in ahp5 background

  • Compare phenotypes and molecular responses to wild-type AHP5

  • Assess effects on interaction with upstream histidine kinases and downstream response regulators

• In vitro reconstitution:

  • Purify components of the phosphorelay system

  • Use antibodies to monitor phosphotransfer from histidine kinases to AHP5

  • Test effects of various factors (e.g., redox conditions, pH, ions) on phosphotransfer efficiency

How might AHP5 antibodies contribute to emerging techniques in plant systems biology?

AHP5 antibodies can enable several cutting-edge approaches in plant systems biology:

• Single-cell proteomics:

  • Use AHP5 antibodies for immunohistochemistry to examine cell-type specific expression

  • Combine with other markers to create protein expression atlases

  • Correlate with single-cell transcriptomic data to identify post-transcriptional regulation

• Protein interaction networks:

  • Implement AHP5 antibodies in high-throughput interaction screens

  • Create comprehensive maps of two-component signaling networks

  • Identify dynamic changes in the interactome under different conditions

• Synthetic biology applications:

  • Engineer modified AHP5 proteins with altered specificity or activity

  • Use antibodies to track expression and function of engineered components

  • Develop biosensors based on AHP5 signaling dynamics

• Computational modeling:

  • Generate quantitative data on AHP5 abundance and modification states

  • Incorporate data into predictive models of plant signaling networks

  • Validate model predictions using antibody-based experimental approaches

What are the key technical specifications of commercially available AHP5 antibodies?

Based on available information about a monoclonal anti-AHP5 antibody :

This antibody was developed as part of a collection representing comprehensive tools for mapping plant stimuli signal transfer from sensor histidine kinase to nuclear response regulators via histidine phosphotransfer proteins .

What genetic resources are available to complement AHP5 antibody studies?

Several genetic resources can be used alongside antibody-based approaches:

These genetic resources, combined with antibody-based approaches, enable comprehensive analysis of AHP5 function in plant signaling networks.

How can researchers design experiments to distinguish the roles of different AHP proteins using antibodies?

To distinguish the specific roles of AHP5 from other AHP proteins, researchers can implement these experimental approaches:

• Combined immunological and genetic approaches:

  • Compare protein expression patterns in wild-type versus various ahp mutant combinations

  • Examine compensatory changes in other AHPs when AHP5 is absent

  • Use antibodies against multiple AHPs to track their relative abundance in different tissues or conditions

• Structure-function analysis:

  • Use domain-specific antibodies to detect functional regions of AHP5

  • Compare conservation of epitopes across different AHP proteins

  • Correlate structural features with functional specificity

• Temporal and spatial expression studies:

  • Compare the expression patterns of different AHPs across development

  • Examine tissue-specific expression using immunohistochemistry

  • Track changes in subcellular localization under different conditions

• Interaction specificity analysis:

  • Identify interacting partners specific to AHP5 versus those shared with other AHPs

  • Use antibodies in competition assays to determine binding preferences

  • Perform quantitative binding studies to compare interaction strengths

Research has shown that despite functional redundancy, AHP5 has distinct characteristics, such as its unique expression pattern in response to abscisic acid and its relatively weak interaction with the CKI1 receiver domain compared to other AHPs .