AUH Antibody

What is AUH protein and why is it an important research target?

AUH (AU RNA binding protein/enoyl-Coenzyme A hydratase) is a bifunctional mitochondrial protein that possesses both RNA-binding and hydratase activities. It plays a critical role in the leucine degradation pathway by catalyzing the hydration of 3-methylglutaconyl-CoA (3-MG-CoA) to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) . Additionally, AUH functions as an RNA-binding protein that recognizes AU-rich elements (AREs) found in the 3' UTRs of rapidly decaying mRNAs including c-fos, c-myc, and granulocyte/macrophage colony stimulating factors . This dual functionality makes AUH an important target for researchers studying mitochondrial metabolism, RNA regulation, and their potential roles in various diseases.

What types of AUH antibodies are commercially available?

Researchers have multiple options when selecting AUH antibodies, including:

• Polyclonal antibodies: These are antibody mixtures derived from different B cell lineages that recognize multiple epitopes of AUH protein. For example, Proteintech offers rabbit polyclonal antibody (17079-1-AP) that reacts with human, mouse, and rat samples .

• Monoclonal antibodies: These are antibodies produced by a single B cell clone that recognize a specific epitope. Abcam provides a rabbit recombinant monoclonal antibody [EPR11087(B)] that reacts with human samples .

The choice between polyclonal and monoclonal antibodies depends on the specific research application, with polyclonals offering broader epitope recognition while monoclonals provide higher specificity for a single epitope.

What are the common applications for AUH antibodies?

AUH antibodies can be utilized in multiple experimental techniques:

For optimal results, researchers should validate these applications with appropriate controls and optimize dilutions for their specific experimental systems .

How should AUH antibodies be stored and handled to maintain reactivity?

To maintain optimal reactivity of AUH antibodies, follow these storage and handling guidelines:

• Store antibodies at -20°C in the recommended buffer (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) .

• Antibodies are generally stable for one year after shipment when stored properly. For the 17079-1-AP antibody, aliquoting is unnecessary for -20°C storage .

• Avoid repeated freeze-thaw cycles, which can lead to antibody degradation and loss of activity.

• When working with antibodies, keep them on ice and return to storage promptly.

• Some antibody preparations contain small amounts of BSA (e.g., 0.1%) which helps stabilize the antibody .

• Follow manufacturer's recommendations for antigen retrieval methods in IHC applications. For AUH antibodies, both TE buffer (pH 9.0) and citrate buffer (pH 6.0) have been successfully used .

What are the recommended protocols for Western blot using AUH antibodies?

For optimal Western blot results with AUH antibodies, follow these methodological guidelines:

• Sample preparation: Extract proteins from tissues or cells using standard lysis buffers containing protease inhibitors. Human brain tissue has shown positive WB results with AUH antibodies .

• Protein separation: Load 10-30 μg of total protein per lane on an SDS-PAGE gel (10-12% recommended).

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard protocols.

• Blocking: Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute AUH antibody according to manufacturer recommendations (1:500-1:3000 for polyclonal antibody 17079-1-AP) . Incubate overnight at 4°C.

• Washing: Wash the membrane 3-5 times with TBST.

• Secondary antibody incubation: Use an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG for most AUH antibodies) and incubate for 1 hour at room temperature.

• Detection: Visualize using ECL substrate and expect to observe a band at approximately 32-33 kDa .

• Controls: Include positive controls such as human fetal kidney, HepG2, or SH-SY5Y lysates, which have demonstrated consistent AUH expression .

How can I optimize immunohistochemistry protocols using AUH antibodies?

To achieve optimal immunohistochemistry results with AUH antibodies, consider these methodological approaches:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissues sectioned at 4-6 μm thickness. Human brain, kidney, heart, and pancreas tissues have demonstrated positive AUH staining .

• Antigen retrieval: This step is critical for AUH detection. Two recommended methods:

  • Heat-mediated antigen retrieval with TE buffer pH 9.0

  • Alternative method using citrate buffer pH 6.0

• Blocking: Block endogenous peroxidase activity with hydrogen peroxide, followed by protein blocking with normal serum.

• Primary antibody incubation: Dilute AUH antibody to 1:20-1:200 for polyclonal antibodies or 1:50 for monoclonal antibodies . Incubate at 4°C overnight or at room temperature for 1-2 hours.

• Detection system: Use an appropriate detection system compatible with the host species of the primary antibody (typically rabbit).

• Counterstaining: Counterstain with hematoxylin for nuclear visualization.

• Controls: Include positive control tissues (brain, kidney, heart) and negative controls (primary antibody omission).

• Evaluation: AUH typically shows cytoplasmic staining pattern consistent with its mitochondrial localization .

How can I use AUH antibodies to investigate the dual functionality of AUH protein?

AUH's unique dual functionality as both an enzyme in metabolic pathways and an RNA-binding protein makes it an intriguing research target. To investigate these distinct functions:

• Metabolic function investigation:

  • Use AUH antibodies in immunoprecipitation followed by activity assays to measure the hydratase activity of immunopurified AUH .

  • Combine with metabolomic approaches to analyze 3-methylglutaconyl-CoA and 3-hydroxy-3-methylglutaryl-CoA levels in cellular systems.

  • Perform co-immunoprecipitation to identify protein interactions within the leucine degradation pathway.

• RNA-binding function investigation:

  • RNA immunoprecipitation (RIP) assays using AUH antibodies can identify the mRNAs bound by AUH in vivo .

  • Cross-linking immunoprecipitation (CLIP) can map the precise binding sites of AUH on target RNAs.

  • Combine with transcriptomic approaches to identify global changes in mRNA stability upon AUH perturbation.

• Integration of dual functions:

  • Design experiments to determine if the metabolic state affects RNA-binding activity or vice versa.

  • Use cellular compartmentalization studies with AUH antibodies to determine if the dual functions occur in distinct subcellular locations.

  • Employ mutational analyses with selective loss of either function, followed by immunological detection using AUH antibodies.

What are the challenges in detecting AUH in different experimental systems?

Researchers face several challenges when detecting AUH across different experimental systems:

• Tissue-specific expression variations:

  • AUH expression levels vary across tissues, with notable expression in brain, kidney, heart, and pancreas .

  • Adjust antibody dilutions and incubation times according to the expected expression level in your tissue of interest.

  • For tissues with lower expression, consider using signal amplification methods or more sensitive detection systems.

• Specificity concerns:

  • Ensure your AUH antibody specifically recognizes AUH and not other proteins with AU-rich binding domains.

  • Validate antibody specificity using knockdown or knockout controls.

  • When studying cross-species samples, confirm the antibody's reactivity with your species of interest (human, mouse, rat) .

• Technical considerations:

  • Antigen retrieval methods significantly impact AUH detection in FFPE tissues; compare TE buffer (pH 9.0) and citrate buffer (pH 6.0) to determine optimal conditions .

  • The bifunctional nature of AUH may result in complex protein-protein or protein-RNA interactions that mask epitopes in certain experimental conditions.

  • Post-translational modifications might affect antibody recognition; consider using multiple antibodies targeting different epitopes.

• Subcellular localization:

  • As a mitochondrial protein, AUH may require specific extraction methods for complete isolation.

  • Consider subcellular fractionation approaches combined with antibody detection to study compartment-specific roles.

How can AUH antibodies be used to study the role of AUH in disease models?

AUH antibodies can be valuable tools for investigating the role of this protein in various disease contexts:

• Neurodegenerative diseases:

  • Use AUH antibodies for immunohistochemical analysis of brain tissues from neurodegenerative disease models to assess changes in expression or localization .

  • Combine with co-localization studies to determine if AUH interacts with disease-relevant proteins.

  • Investigate whether AUH's role in mRNA stability affects the expression of genes implicated in neurodegeneration.

• Metabolic disorders:

  • AUH's role in leucine degradation makes it relevant to inborn errors of metabolism.

  • Use AUH antibodies to compare protein levels and localization in patient-derived samples versus controls.

  • Perform functional assays coupled with immunodetection to correlate protein levels with metabolic abnormalities.

• Cancer research:

  • Analyze AUH expression in tumor versus normal tissues using immunohistochemistry .

  • Investigate whether AUH's RNA-binding function affects the stability of oncogenes or tumor suppressors.

  • Study the relationship between mitochondrial metabolism, AUH function, and cancer progression using antibody-based detection methods.

• Infection and inflammation:

  • AUH may play a role in the C5-dicarboxylate catabolism pathway, which is required to detoxify itaconate, an antimicrobial metabolite produced during infections .

  • Use AUH antibodies to study protein regulation during inflammatory responses.

  • Combine with functional assays to determine how AUH contributes to metabolic adaptations during infection.

Why might I be seeing unexpected bands or staining patterns with my AUH antibody?

When encountering unexpected results with AUH antibodies, consider these potential causes and solutions:

• Multiple bands in Western blot:

  • Expected molecular weight for AUH is 32-33 kDa . Additional bands may represent:

    • Post-translational modifications

    • Alternative splice variants

    • Degradation products

    • Non-specific binding

  • Mitigation strategies:

    • Increase blocking time/concentration

    • Optimize primary antibody dilution (try 1:1000-1:3000 range)

    • Perform more stringent washing steps

    • Include appropriate controls (knockout/knockdown samples)

• Unexpected staining patterns in IHC:

  • AUH typically shows cytoplasmic/mitochondrial staining pattern .

  • Unusual patterns may result from:

    • Cross-reactivity with similar proteins

    • Insufficient antigen retrieval

    • Overfixation of samples

  • Optimization approaches:

    • Compare different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

    • Titrate antibody concentration (1:20-1:200 range is recommended)

    • Use positive control tissues with known AUH expression (brain, kidney, heart)

• High background issues:

  • Could result from:

    • Insufficient blocking

    • Too high antibody concentration

    • Inadequate washing

    • Sample autofluorescence (in IF)

  • Solutions:

    • Extend blocking time or try different blocking agents

    • Increase antibody dilution (e.g., from 1:500 to 1:3000)

    • Add additional/longer washing steps

    • Include appropriate serum from the secondary antibody host species in the blocking buffer

How can I validate the specificity of my AUH antibody?

Validating antibody specificity is critical for reliable experimental results. For AUH antibodies, consider these validation approaches:

• Genetic validation:

  • Perform knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9) of AUH gene

  • Compare Western blot or IHC results between wild-type and AUH-depleted samples

  • Expect significant reduction or absence of signal in depleted samples

• Recombinant protein controls:

  • Use purified recombinant AUH protein as a positive control

  • Perform peptide competition assay by pre-incubating the antibody with the immunizing peptide or recombinant protein

  • Expected result: signal should be substantially reduced after peptide competition

• Orthogonal method validation:

  • Compare antibody results with orthogonal techniques like mass spectrometry

  • Use multiple antibodies targeting different epitopes of AUH

  • Results should be consistent across different detection methods

• Cross-reactivity testing:

  • Test the antibody in tissues/cells known to express AUH (brain, kidney, heart)

  • Include negative controls where AUH expression is minimal

  • Verify that signal intensity correlates with expected expression levels

• Application-specific validation:

  • For Western blot: confirm band at correct molecular weight (32-33 kDa)

  • For IHC/IF: verify expected subcellular localization (mitochondrial/cytoplasmic)

  • For IP: confirm enrichment of AUH protein in immunoprecipitated fraction

What are the considerations for using AUH antibodies in RNA immunoprecipitation (RIP) assays?

RNA immunoprecipitation is a valuable technique for studying RNA-binding proteins like AUH. When using AUH antibodies for RIP assays, consider these methodological aspects:

• Experimental design considerations:

  • Cross-linking: Decide whether to use formaldehyde cross-linking to stabilize protein-RNA interactions

  • Cell/tissue preparation: Optimize lysis conditions to maintain RNA integrity while efficiently extracting AUH

  • Controls: Include IgG control, input samples, and potentially AUH-depleted samples

• Antibody selection and optimization:

  • Select antibodies validated for immunoprecipitation applications

  • Determine optimal antibody amount (recommended range: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Consider testing multiple AUH antibodies recognizing different epitopes

• Protocol optimization:

  • RNase inhibitors: Include RNase inhibitors in all buffers to prevent RNA degradation

  • Washing stringency: Balance between removing non-specific interactions and maintaining specific AUH-RNA complexes

  • Elution conditions: Optimize to efficiently release AUH-RNA complexes without degrading RNA

• Downstream analysis considerations:

  • RNA extraction: Use methods that yield high-quality RNA from potentially small amounts

  • Detection methods: Consider RT-qPCR for specific targets, microarray, or RNA-seq for global analysis

  • Data analysis: Compare enrichment to input and IgG controls; identify significantly enriched RNAs

• Validation of RIP results:

  • Confirm AUH binding to identified RNAs using orthogonal methods (e.g., EMSA, RNA pull-down)

  • Analyze identified RNAs for presence of AU-rich elements

  • Investigate functional consequences of AUH binding on mRNA stability or translation

How can AUH antibodies be used in studying the connection between mitochondrial metabolism and RNA regulation?

AUH's dual functionality provides a unique opportunity to investigate the interplay between metabolism and RNA regulation:

• Subcellular co-localization studies:

  • Use AUH antibodies in combination with mitochondrial markers and RNA visualization techniques

  • Determine whether AUH's RNA binding activity occurs within mitochondria or in other cellular compartments

  • Investigate whether metabolic state affects AUH localization and RNA-binding capacity

• Metabolic perturbation experiments:

  • Apply metabolic stressors (e.g., leucine starvation, mitochondrial inhibitors)

  • Use AUH antibodies to track changes in protein localization, expression, or post-translational modifications

  • Correlate these changes with alterations in target mRNA stability

• Immunoprecipitation-based approaches:

  • Perform sequential IP-RIP experiments to identify metabolic enzymes that interact with AUH and determine their effect on RNA binding

  • Use AUH antibodies to immunoprecipitate protein complexes under different metabolic conditions

  • Identify metabolic intermediates associated with AUH-RNA complexes

• Disease model applications:

  • Investigate diseases with both metabolic and RNA regulation components

  • Use AUH antibodies to examine whether pathological conditions affect the balance between AUH's dual functions

  • Determine if therapeutic interventions targeting metabolism influence AUH's RNA-binding activity

What are the considerations for using AUH antibodies in multi-omics research approaches?

Integrating AUH antibody-based techniques with multi-omics approaches can provide comprehensive insights:

• Integration with proteomics:

  • Use AUH antibodies for immunoprecipitation followed by mass spectrometry to identify protein interaction networks

  • Compare AUH protein complexes under different cellular conditions

  • Investigate post-translational modifications that might regulate AUH's dual functionality

• Integration with transcriptomics:

  • Combine RIP using AUH antibodies with RNA-seq to identify the global RNA targets of AUH

  • Correlate changes in AUH expression or localization with transcriptome-wide alterations

  • Analyze the structural and sequence features of AUH-bound RNAs

• Integration with metabolomics:

  • Use AUH antibodies to manipulate AUH levels (through immunodepletion or overexpression)

  • Analyze resultant changes in metabolite profiles, particularly in the leucine degradation pathway

  • Correlate metabolic alterations with changes in RNA stability of AUH targets

• Spatial multi-omics considerations:

  • Use AUH antibodies for spatial proteomics to determine subcellular localization patterns

  • Combine with spatial transcriptomics to analyze co-localization of AUH with its RNA targets

  • Integrate spatial metabolomics to understand the relationship between local metabolite concentrations and AUH function

• Data integration challenges:

  • Develop computational frameworks to integrate antibody-based localization/interaction data with other omics datasets

  • Account for technical variations in antibody performance when comparing across datasets

  • Establish causal relationships between AUH's metabolic and RNA-binding functions