ADR1 Antibody

What is ADR1 and what cellular functions does it regulate?

ADR1 refers to multiple distinct proteins across different species, each with unique functions that require careful consideration when designing antibody-based experiments. In humans, ADRM1 (Adhesion Regulating Molecule 1, also known as ARM-1) functions as a component of the 26S proteasome complex involved in ATP-dependent degradation of ubiquitinated proteins . This complex plays a critical role in protein homeostasis by removing misfolded or damaged proteins that could impair cellular functions . Within this system, ADRM1 specifically functions as a proteasomal ubiquitin receptor that engages and activates 19S-associated deubiquitinases UCHL5 and PSMD14 during protein degradation .

In contrast, ADR-1 in C. elegans is a dsRNA binding protein that regulates adenosine-to-inosine (A-to-I) RNA editing levels . In yeast, Adr1 functions as a zinc-finger transcriptional activator that regulates glucose-responsive genes . When designing experiments using ADR1 antibodies, researchers must clearly identify which specific ADR1 protein their studies target to ensure appropriate experimental controls and interpretation.

How do I select the appropriate ADR1 antibody for my specific experiment?

When selecting an ADR1 antibody, consider the following methodological factors:

• Target specificity: Determine which ADR1 homolog you're investigating (human ADRM1/ARM-1, C. elegans ADR-1, or yeast Adr1) and select an antibody raised against that specific protein .

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, immunohistochemistry, immunoprecipitation, etc.). For instance, the rabbit polyclonal ADRM1/ARM-1 antibody (ab236774) has been validated for Western blot (WB), immunohistochemistry-paraffin (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) applications .

• Species cross-reactivity: Confirm the antibody recognizes your target species. For example, ab236774 has been shown to react with both human and mouse ADRM1/ARM-1 .

• Immunogen information: Review the immunogen used to generate the antibody. For ab236774, the immunogen corresponds to the recombinant full-length human ADRM1 protein .

• Validation data: Request and review validation data showing the antibody's specificity, such as Western blots demonstrating a single band of the expected molecular weight and appropriate negative controls.

What are the recommended protocols for validating ADR1 antibody specificity?

A rigorous validation protocol for ADR1 antibodies should include:

• Positive and negative controls: Include lysates from tissues or cells known to express or not express your target ADR1 protein. For ADRM1 validation, compare reactivity in wild-type samples versus ADRM1 knockout or knockdown samples.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application. Specific binding should be blocked by the peptide, as demonstrated in the phospho-specific Adr1 antibody validation where a phosphorylated peptide efficiently competed with protein detection while a non-phosphorylated peptide did not .

• Multiple antibody approach: Use antibodies raised against different epitopes of the same protein and compare detection patterns.

• Immunoprecipitation followed by mass spectrometry: This technique can confirm that the antibody is capturing the intended target protein.

• Tagged protein expression: Compare antibody recognition with detection via an epitope tag, as demonstrated in yeast studies where 3x FLAG-tagged ADR-1 was expressed and immunoprecipitated to validate specificity .

How can I optimize Western blot conditions for ADR1 antibody detection?

For optimal Western blot detection of ADR1 proteins:

• Buffer selection: For human ADRM1/ARM-1, standard RIPA or NP-40 lysis buffers are typically effective. For yeast Adr1, specialized extraction methods as described by Horvath and Reizman or the rapid yeast protein prep method have been successfully employed .

• Protein separation: For ADRM1 (110 kDa), use 8-10% SDS-PAGE gels or gradient gels (4-12%). For larger phosphorylated proteins like yeast Adr1, Tris-Acetate 3-8% gels have been effective for resolving phosphorylation-induced mobility shifts .

• Transfer conditions: Use wet transfer methods with 10-20% methanol for larger proteins. For Adr1 in particular, transfer at lower voltage (30V) overnight at 4°C may improve transfer efficiency of larger proteins.

• Blocking optimization: Test both BSA and milk-based blocking buffers, as some phospho-specific antibodies (like those against phosphorylated Adr1) may be inhibited by phosphoproteins in milk.

• Antibody dilution: Start with manufacturer recommendations (typically 1:500-1:1000 for Adr1-specific antibodies) and optimize as needed .

• Detection system: For quantitative analysis, consider infrared-based detection systems as used for Adr1, which allow for more accurate quantification than chemiluminescence .

How can ADR1 antibodies be used to investigate protein-protein interactions within the proteasome complex?

ADR1 antibodies can effectively elucidate protein-protein interactions within multiprotein complexes through several advanced techniques:

• Co-immunoprecipitation (Co-IP): ADR1 antibodies can immunoprecipitate the target protein along with its interacting partners. For human ADRM1, this approach has been used to study its interactions with deubiquitinases UCHL5 and PSMD14 within the 19S regulatory particle of the proteasome . The protocol should include:

  • Cross-linking (optional but recommended for transient interactions)

  • Cell lysis in non-denaturing conditions

  • Pre-clearing lysate with protein A/G beads

  • Immunoprecipitation with ADR1 antibody

  • Washing under conditions that maintain protein-protein interactions

  • Western blot analysis of co-precipitated proteins

• Proximity ligation assay (PLA): This technique can visualize and quantify protein-protein interactions in situ with single-molecule resolution, providing spatial information about where ADRM1 interacts with its partners within the cell.

• RNA immunoprecipitation (RIP): For RNA-binding proteins like C. elegans ADR-1, RIP assays using epitope-tagged ADR-1 have demonstrated that ADR-1 directly binds to known editing targets in vivo and that ADR-1 and ADR-2 co-occupy transcripts . This approach revealed that binding of ADR-1 to mRNAs is independent of ADR-2, as mRNAs were enriched to a similar extent in FLAG-ADR-1 immunoprecipitates regardless of ADR-2 presence .

What methodologies can address discrepancies in ADR1 antibody detection between different experimental approaches?

When encountering discrepancies in ADR1 antibody detection across different experimental methods:

• Epitope accessibility analysis: Different experimental conditions may affect epitope accessibility. For phosphorylation-specific antibodies like those against phosphorylated Ser98 in yeast Adr1, buffer conditions and sample preparation methods can significantly impact detection . Compare native versus denaturing conditions and test different fixation methods for immunocytochemistry.

• Post-translational modification consideration: For proteins like yeast Adr1 where phosphorylation regulates function, use phosphatase treatments to determine if discrepancies result from different phosphorylation states . Include phosphatase inhibitors (sodium pyrophosphate, sodium orthovanadate, β-glycerophosphate, EGTA, and sodium fluoride) when attempting to preserve phosphorylation states .

• Cross-validation with orthogonal methods: Combine antibody-based detection with mass spectrometry or functional assays. For yeast Adr1, researchers confirmed phosphorylation at Ser98 using both antibody detection and in vitro phosphorylation followed by tryptic peptide mapping and phosphoamino acid analysis .

• Controlled expression systems: Use tagged versions of the protein expressed at defined levels to establish a standard curve for antibody sensitivity, as demonstrated with FLAG-tagged ADR-1 in C. elegans studies .

How can phospho-specific ADR1 antibodies be used to investigate regulatory mechanisms?

Phospho-specific antibodies against ADR1 proteins provide valuable tools for studying regulatory mechanisms:

• Temporal dynamics of phosphorylation: For yeast Adr1, phospho-specific antibodies targeting Ser98 revealed that phosphorylation status changes in response to glucose levels, providing insight into how post-translational modification regulates this transcriptional activator . Researchers can use time-course experiments with these antibodies to track phosphorylation changes during cellular responses.

• Screening for regulatory kinases: The phospho-specific antibody against Ser98 of yeast Adr1 was used in a screen of 102 yeast kinase deletion strains to identify kinases responsible for Adr1 phosphorylation . This methodological approach can be adapted to identify kinases for other ADR1 proteins by:

  • Expressing the ADR1 protein in wild-type and kinase-deletion/inhibited backgrounds

  • Analyzing phosphorylation status with phospho-specific antibodies

  • Confirming direct phosphorylation with in vitro kinase assays

• Structure-function analysis: Combine phospho-specific antibody detection with mutational analysis (e.g., phosphomimetic S98D and non-phosphorylatable S98A mutations in yeast Adr1) to dissect the functional consequences of phosphorylation . This approach revealed that phosphorylation proximal to the zinc-finger domain affects DNA binding activity.

• Subcellular localization studies: Use phospho-specific antibodies in immunofluorescence microscopy to determine if phosphorylation affects the subcellular localization of ADR1 proteins, providing insight into regulatory mechanisms.

What experimental approaches can resolve contradictory findings regarding ADR1 function across different model systems?

When addressing contradictory findings regarding ADR1 function across different experimental models:

How can I address non-specific binding when using ADR1 antibodies?

To minimize non-specific binding with ADR1 antibodies:

• Antibody titration: Optimize antibody concentration through systematic dilution series. For Adr1-specific antibodies, concentrations ranging from 1:500 to 1:3000 have been successful depending on the application .

• Blocking optimization: Test different blocking agents (BSA, casein, commercial blocking buffers) and concentrations. For phospho-specific antibodies like those against Adr1, milk-based blockers may interfere with recognition due to phosphoproteins in milk .

• Extended washing protocols: Implement more stringent washing steps, particularly for immunoprecipitation experiments with ADR1 antibodies. For in vitro phosphorylation studies of Adr1, researchers used four washes in buffer containing 300mM NaCl, 10mM imidazole, and 0.005% Tween 20 to remove non-specific interactions .

• Pre-adsorption: For polyclonal antibodies, pre-adsorb against lysates from cells lacking the target protein. This approach is particularly useful for C. elegans studies where comparing immunoprecipitates from FLAG-ADR-1 and adr-1(-) worms helped establish binding specificity .

• Peptide competition: Include a gradient of immunizing peptide to confirm specificity, as demonstrated with the phospho-specific Adr1 antibody where a phosphorylated peptide competed for antibody binding but the non-phosphorylated peptide did not .

What controls are essential when using ADR1 antibodies for quantitative analysis of protein expression?

For rigorous quantitative analysis with ADR1 antibodies:

• Loading controls: Include housekeeping proteins (β-actin, GAPDH) or total protein stains (Ponceau S, SYPRO Ruby) to normalize for loading variations.

• Standard curve: Generate a standard curve using recombinant protein or cell lysates with known quantities of the target protein to ensure detection is in the linear range.

• Biological replicates: Include at least three biological replicates to account for natural variation in protein expression.

• Technical replicates: Perform technical replicates of each Western blot to account for technical variation in transfer efficiency and antibody binding.

• Knockout/knockdown controls: Include samples from knockout or knockdown experiments to establish the specificity of the antibody signal.

• Internal reference samples: Include a common reference sample across all blots when comparing multiple experimental conditions that cannot fit on a single gel.

• Quantification method controls: For fluorescent-based detection systems like those used for Adr1, ensure that secondary antibodies are used at concentrations that do not cause signal saturation .

How can I optimize immunoprecipitation protocols for ADR1 proteins that are difficult to extract or detect?

For challenging ADR1 immunoprecipitation experiments:

• Extraction buffer optimization: For membrane-associated proteins like human ADRM1 (originally identified as a cell membrane glycoprotein), test different detergent combinations (CHAPS, digitonin, DDM) that preserve protein-protein interactions while effectively solubilizing membrane proteins .

• Cross-linking approach: Implement reversible cross-linking (DSP, formaldehyde) before cell lysis to capture transient interactions or stabilize complexes. This is particularly useful for studying ADRM1's interactions within the proteasome complex .

• Tagged protein expression: When endogenous protein detection is challenging, express tagged versions of the protein. For C. elegans ADR-1, researchers used 3x FLAG-tagged ADR-1 for immunoprecipitation when a polyclonal antibody was inefficient .

• Bead selection: Compare different affinity matrices (protein A/G, magnetic beads) for optimal capture efficiency. For in vitro phosphorylation studies of Adr1, Ni-NTA magnetic agarose beads provided effective capture through the His6 tag .

• Elution optimization: For ADR1 proteins that are difficult to elute, compare different elution methods (pH, ionic strength, competitive elution with peptides, SDS elution). For His-tagged Adr1, boiling in LDS loading buffer provided effective elution after in vitro phosphorylation .

What are the best practices for using ADR1 antibodies in multi-color immunofluorescence experiments?

For successful multi-color immunofluorescence with ADR1 antibodies:

• Antibody species selection: Choose primary antibodies raised in different host species to avoid cross-reactivity. When studying proteasome components, pair rabbit polyclonal ADRM1/ARM-1 antibodies with mouse monoclonals against other proteasome subunits .

• Sequential staining protocol: For antibodies raised in the same species, use sequential staining with intermediate blocking steps using excess unconjugated Fab fragments against the first primary antibody.

• Controls for spectral overlap: Include single-color controls to establish compensation settings for fluorescence microscopy or flow cytometry.

• Peptide competition controls: Include wells treated with antibody pre-incubated with immunizing peptide to establish specificity of staining.

• Cross-adsorbed secondary antibodies: Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity between detection systems.

• Fixation optimization: Compare different fixation methods (paraformaldehyde, methanol, acetone) as epitope accessibility for ADR1 proteins may be affected by fixation method.

• Signal amplification: For low-abundance ADR1 proteins, consider tyramide signal amplification or other amplification methods to enhance detection sensitivity.

How can ADR1 antibodies be utilized in studying proteasome dynamics in neurodegenerative diseases?

ADR1 antibodies offer valuable tools for investigating proteasome dysfunction in neurodegenerative conditions:

• Quantitative analysis of ADRM1 levels: Use validated antibodies to compare ADRM1 expression between healthy and diseased tissues. Human ADRM1/ARM-1 antibodies can be applied to patient-derived samples to assess whether proteasome receptor abundance changes correlate with disease progression .

• Co-localization with protein aggregates: Implement dual immunofluorescence to determine if ADRM1 co-localizes with disease-specific protein aggregates (e.g., amyloid plaques, tau tangles, α-synuclein inclusions). This approach can reveal whether the ubiquitin receptor component of the proteasome is recruited to these pathological structures.

• Assessment of proteasome integrity: Use ADRM1 antibodies alongside antibodies against other proteasome components to evaluate whether proteasome complexes remain intact in disease states. Changes in co-immunoprecipitation efficiency could indicate structural alterations in the proteasome machinery.

• In situ proximity ligation assays: Combine ADRM1 antibodies with antibodies against deubiquitinases (UCHL5, PSMD14) to visualize and quantify their interactions in diseased versus healthy neurons, potentially revealing dysfunction in the ubiquitin-proteasome pathway .

• Live-cell imaging: Correlate antibody-based findings in fixed cells with live-cell studies using fluorescently-tagged ADRM1 to track proteasome dynamics in response to proteotoxic stress.

What methodological approaches can integrate ADR1 antibody-based detection with RNA editing analysis?

To effectively combine protein detection with RNA editing assessment:

• Sequential RNA-protein isolation: Develop protocols that allow extraction of both RNA and protein from the same sample, enabling direct correlation between ADR-1 protein levels and editing efficiency in C. elegans models .

• RNA immunoprecipitation followed by sequencing (RIP-seq): Optimize RNA immunoprecipitation protocols using ADR-1 antibodies followed by high-throughput sequencing to identify all RNA targets, as demonstrated in C. elegans where this approach identified 48 novel edited transcripts .

• Correlative microscopy: Combine immunofluorescence detection of ADR-1 with RNA FISH (fluorescence in situ hybridization) to visualize the spatial relationship between the protein and its target RNAs within cells.

• Integrated analysis pipeline: Develop computational tools that correlate RNA editing sites identified by sequencing with ADR-1 binding sites identified by techniques such as CLIP (cross-linking immunoprecipitation) using ADR-1 antibodies.

• Structure-function analysis: Use antibodies against wild-type and mutant ADR-1 proteins (e.g., dsRNA binding domain mutants) to assess how structural alterations affect both protein expression and RNA target recognition .

How can phosphoproteomics be integrated with ADR1 antibody-based detection to map regulatory networks?

Integrating phosphoproteomics with antibody-based detection provides a comprehensive view of ADR1 regulation:

• Quantitative phosphoproteomics workflow: Combine phospho-specific antibodies against ADR1 proteins with mass spectrometry-based phosphopeptide enrichment to identify all phosphorylation sites. For yeast Adr1, this approach could extend beyond the characterized Ser98 site to map the complete phosphorylation landscape .

• Temporal phosphorylation dynamics: Use phospho-specific antibodies to track changes in ADR1 phosphorylation status over time following specific stimuli (e.g., glucose availability for yeast Adr1) . Correlate these changes with global phosphoproteome alterations to identify coordinated regulatory events.

• Kinase-substrate relationship mapping: Combine in vitro kinase assays using purified ADR1 proteins with validation in cellular contexts using phospho-specific antibodies. This approach was used for yeast Adr1 to identify kinases responsible for Ser98 phosphorylation .

• Phosphorylation site-specific functional analysis: Generate phosphomimetic and non-phosphorylatable mutants (as done with S98A and S98D in yeast Adr1) and use antibodies to confirm expression levels when comparing functional outcomes .

• Computational integration: Develop algorithms that integrate antibody-based quantification of phosphorylation states with global phosphoproteomic datasets to predict regulatory networks controlling ADR1 function.

What are the considerations for developing ADR1 antibodies against post-translationally modified forms beyond phosphorylation?

Developing antibodies against novel post-translational modifications (PTMs) of ADR1 proteins requires:

• PTM prediction and validation: Use computational tools to predict potential sites for ubiquitination, acetylation, methylation, or SUMOylation on ADR1 proteins, then validate these sites using mass spectrometry before antibody production.

• Immunogen design strategies: For human ADRM1, which functions within the ubiquitin-proteasome system, antibodies against ubiquitinated forms may require branched peptides that incorporate the ubiquitin remnant (K-ε-GG) at specific lysine residues .

• Validation controls: Generate mutants where the modified residue is replaced with a non-modifiable amino acid. For example, lysine to arginine mutations can prevent ubiquitination while maintaining charge properties, providing essential negative controls for antibody validation.

• Enrichment prior to detection: Incorporate enrichment steps (e.g., ubiquitin remnant motif immunoaffinity purification) before Western blot analysis with ADR1 antibodies to increase sensitivity for low-abundance modified forms.

• Cross-reactivity assessment: Thoroughly evaluate antibodies against modified ADR1 for potential cross-reactivity with other proteins carrying similar modifications, particularly within the same protein family or functional complex.