AMSH2 Antibody

What is AMSH2 and what biological functions does it serve?

AMSH2 belongs to a family of deubiquitinating enzymes that includes AMSH and AMSH3. It functions as a positive regulator of TGF-β signaling by negating the inhibitory effect of Smad7. AMSH2 interacts with inhibitory Smads (particularly Smad7) and potentially with Smad2, but shows little to no interaction with Smads 3 and 4 . Northern blot analysis reveals that AMSH2 mRNA is widely expressed across multiple human tissues and cell lines, including brain, testes, bone, pancreas, liver, kidney, and various cancer cell lines .

The protein is approximately 56 kDa, which aligns with its predicted molecular weight. Its expression has been confirmed in several cell lines, including HepG2 and 293T cells . In plants, AMSH2 possesses deubiquitinase (DUB) activity and is essential for plant viability, as homozygous AMSH2 knockouts are lethal .

What are the key differences between AMSH2 and other AMSH family members?

Although AMSH2 shares significant homology with AMSH and AMSH3, there are functional differences:

Unlike AMSH, AMSH2 shows interaction with Smad2 (an R-Smad), suggesting potentially distinct regulatory mechanisms . In Arabidopsis, AMSH2 does not interact with ALIX in yeast two-hybrid screens, whereas AMSH1 and AMSH3 do .

What criteria should be considered when selecting an AMSH2 antibody for research?

When selecting an AMSH2 antibody, researchers should consider:

• Target specificity: Ensure the antibody specifically recognizes AMSH2 and not other AMSH family members, particularly given their sequence homology.

• Application validation: Verify the antibody has been validated for your specific application (WB, IP, IF, IHC).

• Species reactivity: Confirm reactivity with your species of interest.

• Epitope information: Understand which region of AMSH2 the antibody targets to ensure it's accessible in your experimental conditions.

• Validation methods used: Prioritize antibodies validated using knockout controls, which is considered the gold standard for antibody validation .

• Renewable source: Consider monoclonal or recombinant antibodies over polyclonal ones for better reproducibility .

• Independent validation: Check if the antibody has been validated by independent groups or third-party validators like YCharOS .

How should I validate an AMSH2 antibody before using it in my experiments?

A comprehensive validation approach should include:

• Knockout/knockdown controls: Test the antibody on samples with and without AMSH2 expression (CRISPR knockout cell lines are ideal) .

• Orthogonal validation: Compare antibody results with an antibody-independent method like mass spectrometry or mRNA expression data.

• Multiple antibodies approach: Test several antibodies targeting different epitopes of AMSH2.

• Overexpression controls: Test the antibody on samples overexpressing AMSH2.

• Application-specific validation: Validate the antibody specifically for each application you intend to use it for, as performance can vary between applications.

• Blocking peptide experiment: If available, use the immunogen peptide to block the antibody and confirm specificity.

Analysis by Ayoubi et al. (2023) found that when tested across multiple applications, only about 44% of antibodies recommended for Western blot were actually successful, and of those recommended for immunoprecipitation, only 58% enriched their cognate target from cell extracts . This emphasizes the importance of thorough validation.

What are the optimal conditions for using AMSH2 antibodies in Western blot applications?

Optimal Western blot conditions for AMSH2 antibodies include:

• Sample preparation:

  • For cellular AMSH2: Use RIPA buffer with protease inhibitors for cell lysis

  • Expected band size: ~56 kDa based on immunoprecipitation studies

• Gel separation:

  • 10% SDS-PAGE is generally suitable for resolving proteins in the 50-60 kDa range

• Transfer conditions:

  • PVDF membranes often provide better results for detecting proteins in this molecular weight range

• Blocking and antibody incubation:

  • Start with 1:1000 to 1:2000 dilution (based on similar antibodies like AMSH-LP )

  • Include appropriate negative controls (non-expressing cells or AMSH2 knockouts)

  • Include positive controls (cells known to express AMSH2, such as HepG2 or 293T cells)

• Detection system:

  • Enhanced chemiluminescence is generally suitable

  • For low abundance targets, consider more sensitive detection methods

How can I optimize immunoprecipitation protocols using AMSH2 antibodies?

For successful immunoprecipitation of AMSH2:

• Lysis conditions:

  • Use non-denaturing lysis buffers (e.g., 150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, 1% NP-40) to preserve protein-protein interactions

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation states

• Pre-clearing:

  • Pre-clear lysates with appropriate control beads to reduce non-specific binding

• Antibody amount:

  • Start with 2-5 μg of antibody per mg of total protein

  • Titrate to determine optimal concentration

• Controls:

  • Include an isotype control antibody IP

  • If possible, include AMSH2-knockout cells as negative control

• Validation of IP:

  • Confirm successful IP by Western blot using a different AMSH2 antibody targeting a different epitope

  • Consider mass spectrometry analysis of immunoprecipitated samples to confirm identity and identify interacting partners

• Co-IP considerations:

  • When studying interactions with Smads or other partners, gentler washing conditions may help preserve interactions

  • For studying AMSH2-Smad7 interactions, reference the protocols used by Ibarrola et al. (2004)

Why might Western blot with AMSH2 antibodies show multiple bands or unexpected molecular weights?

Multiple bands or unexpected molecular weights can occur for several reasons:

• Post-translational modifications: AMSH2 may undergo phosphorylation, ubiquitination, or other modifications that alter migration.

• Splice variants: Check databases for known splice variants of AMSH2 that could result in different molecular weights.

• Cross-reactivity: The antibody may be cross-reacting with other AMSH family members (AMSH, AMSH3) or unrelated proteins. Validation using knockout controls is crucial to identify true AMSH2 bands.

• Protein degradation: Ensure proper sample handling and include protease inhibitors to prevent degradation products.

• Non-specific binding: Optimize blocking conditions and antibody concentration to reduce background.

• Antibody quality issues: Even monoclonal antibodies can demonstrate non-specific binding. Studies have shown that up to 35% of monoclonal antibody preparations can have staining patterns unrelated to their antigenic specificity .

If experiencing multiple bands, validation using siRNA knockdown or CRISPR knockout of AMSH2 would help identify which band represents the true target protein.

How can I differentiate between AMSH2 and other AMSH family members in my experiments?

Differentiating between AMSH family members requires careful experimental design:

• Antibody selection: Choose antibodies raised against regions with the least homology between family members. Consult sequence alignments to identify unique regions of AMSH2.

• Knockout/knockdown controls: Use specific knockouts or knockdowns of each family member to determine antibody specificity.

• Molecular weight discrimination: Note that human AMSH, AMSH2, and AMSH3 have slightly different molecular weights, which might help distinguish them on Western blots.

• Functional assays: Utilize the different functional properties:

  • AMSH2 particularly affects TGF-β signaling and interacts with Smad7

  • AMSH demonstrates stronger interaction with Smad6

  • Testing effects on different signaling pathways may help identify which family member is active

• Mass spectrometry: Use targeted MS approaches to identify unique peptides specific to each family member.

How can AMSH2 antibodies be used to study its role in TGF-β signaling pathways?

AMSH2 antibodies can be applied in several advanced approaches to study TGF-β signaling:

• Co-immunoprecipitation studies: Use AMSH2 antibodies to pull down protein complexes and identify interacting partners in TGF-β signaling. This has successfully identified interactions between AMSH2 and Smad7 .

• Chromatin immunoprecipitation (ChIP): If AMSH2 is involved in transcriptional regulation through Smads, ChIP can identify DNA binding sites.

• Proximity ligation assays (PLA): Detect and visualize protein-protein interactions between AMSH2 and Smad proteins in situ.

• Immunofluorescence co-localization: Track changes in AMSH2 subcellular localization in response to TGF-β stimulation.

• Phosphorylation-specific antibodies: If AMSH2 is regulated by phosphorylation, phospho-specific antibodies could track its activation state.

• Activity-based probes: Combine with activity-based probes to monitor the DUB activity of AMSH2 in different cellular contexts.

• FRET/BRET approaches: When combined with appropriate tags, can monitor real-time interactions between AMSH2 and components of the TGF-β pathway.

The key experiment by Ibarrola et al. demonstrated that overexpression of AMSH2 increased luciferase activity driven by the 3TP promoter upon TGF-β stimulation, while siRNA knockdown of AMSH2 decreased this activity .

What methods are available to study the deubiquitinating enzyme activity of AMSH2 using specific antibodies?

To study AMSH2's deubiquitinating enzyme activity:

• In vitro DUB assays:

  • Immunoprecipitate AMSH2 using validated antibodies

  • Incubate with fluorescently labeled di-ubiquitin substrates (like di-ubiquitin-TAMRA)

  • Monitor cleavage through increased fluorescence as demonstrated with AMSH3

• Cellular ubiquitination profiles:

  • Compare ubiquitination patterns in cells with and without AMSH2 (using knockdown/knockout)

  • Immunoprecipitate potential substrates and blot for ubiquitin

  • Focus on Lys63-linked ubiquitin chains, which are preferred by related AMSH family members

• DUB activity probes:

  • Use ubiquitin-based activity probes that covalently bind active DUBs

  • Immunoprecipitate with AMSH2 antibodies and analyze probe binding

• Substrate identification:

  • Combine AMSH2 immunoprecipitation with mass spectrometry to identify potential substrates

  • Validate candidates by monitoring their ubiquitination status after AMSH2 modulation

• Structure-function studies:

  • Use AMSH2 antibodies to validate expression of catalytic mutants

  • Compare the impact of wild-type vs. catalytically inactive AMSH2 on cellular phenotypes

For example, when studying AMSH3 in Arabidopsis, researchers found that ALIX protein doesn't influence the DUB activity of AMSH3 in vitro using fluorescent di-ubiquitin substrates . Similar approaches could be applied to human AMSH2.

How are new technologies improving the specificity and application range of AMSH2 antibodies?

Several emerging technologies are enhancing AMSH2 antibody research:

• Recombinant antibody technologies: Moving from polyclonal to recombinant antibodies improves batch-to-batch reproducibility. Studies show recombinant antibodies demonstrate greater effectiveness and reproducibility than polyclonal antibodies .

• Library-on-library approaches: New screening methods allow many antigens to be probed against many antibodies simultaneously, identifying specific interacting pairs with greater efficiency .

• Active learning for antibody optimization: Machine learning models can predict antibody-antigen binding and guide the improvement of specificity. Some algorithms have reduced the number of required antigen mutant variants by up to 35% .

• Nanobodies and single-domain antibodies: These smaller antibody fragments can access epitopes that conventional antibodies cannot reach, potentially enabling more specific AMSH2 detection.

• CRISPR-based validation platforms: High-throughput generation of knockout cell lines enables more robust antibody validation. Standardized approaches using parental and knockout cell lines have become the gold standard for validating antibody specificity .

• Multiplexed antibody validation: Techniques like cyclic immunofluorescence allow simultaneous testing of multiple antibodies against the same sample, improving comparative validation.

• Engineered specificity: Computational design of antibodies with customized specificity profiles can generate reagents with either highly specific binding to AMSH2 or cross-specificity for multiple AMSH family members based on research needs .

What are the considerations for developing phospho-specific AMSH2 antibodies?

Developing phospho-specific AMSH2 antibodies requires:

• Phosphorylation site identification:

  • Use mass spectrometry to identify physiologically relevant phosphorylation sites on AMSH2

  • Prioritize sites that change in response to TGF-β stimulation or other relevant stimuli

• Immunogen design:

  • Create phosphopeptides corresponding to identified phosphorylation sites

  • Include carrier proteins to enhance immunogenicity

  • Consider multiple adjacent phosphorylation sites if they occur together

• Validation strategy:

  • Test antibodies against wild-type AMSH2 and phospho-site mutants (S/T→A or Y→F)

  • Validate with phosphatase-treated samples (should eliminate signal)

  • Demonstrate signal changes after relevant cellular stimulation

• Specificity considerations:

  • Cross-check against similar phosphorylation motifs in related proteins

  • Perform blocking peptide experiments with both phosphorylated and non-phosphorylated peptides

  • Validate using knockout controls treated with appropriate stimuli

• Application optimization:

  • Include phosphatase inhibitors during sample preparation

  • Optimize fixation conditions for immunofluorescence to preserve phospho-epitopes

  • Consider rapid sample processing to capture transient phosphorylation events

Phospho-specific antibodies require another level of specificity and validation compared to standard antibodies , and both the phosphorylated and non-phosphorylated forms should be included in validation studies.

How can researchers address batch-to-batch variability in AMSH2 antibody performance?

To address batch-to-batch variability:

• Select recombinant or monoclonal antibodies: These offer better reproducibility than polyclonal antibodies. Data from large-scale validation studies indicate that monoclonal antibodies show greater consistency .

• Maintain reference samples: Keep aliquots of positive control samples from successful experiments to test new antibody batches.

• Detailed record keeping: Document lot numbers, dilutions, and exact protocols that worked successfully.

• Standardized validation protocols: Implement consistent validation protocols for each new batch:

  • Western blot on known AMSH2-expressing cell lines

  • Knockout/knockdown controls

  • Compare signal intensity and pattern to previous batches

• Request certificate of analysis: When purchasing, request detailed validation data specific to the lot being purchased.

• Internal controls: Include loading controls and positive controls in every experiment to normalize for technical variation.

• Consider antibody pooling: For critical experiments, pooling antibodies from multiple manufacturing batches may reduce batch-specific biases.

Studies have shown significant variability between antibody lots, with one study reporting an R² value of just 0.038 between different lots of the same antibody .

What are the international standards and guidelines for validation of antibodies like those against AMSH2?

International standards and guidelines for antibody validation include:

• The five pillars approach: Proposed by the International Working Group for Antibody Validation (2016), it recommends using at least one of these strategies :

  • Genetic strategies (knockout/knockdown)

  • Orthogonal strategies (comparing antibody results with antibody-independent methods)

  • Multiple antibody strategies (using different antibodies against the same target)

  • Recombinant expression strategies (expression of tagged proteins)

  • Immunocapture mass spectrometry (identifying proteins captured by the antibody)

• RRID (Research Resource Identifier) program: A system for standardized referencing of antibodies in publications, improving tracking and reproducibility .

• YCharOS guidelines: This independent antibody characterization organization emphasizes standardized testing using knockout cell lines .

• GBSI (Global Biological Standards Institute) recommendations: Focus on transparency in reporting antibody validation methods and results.

• Independent validation repositories: Resources like Antibodypedia and CiteAb aggregate validation data from multiple sources.

• FDA guidance for analytical method validation: Defines validation as "the process of demonstrating, through the use of specific laboratory investigations, that the performance characteristics of an analytical method are suitable for its intended analytical use" .

• Application-specific validation: Different criteria exist for different applications (WB, IP, IF, IHC, etc.). For instance, antibodies successful in Western blot may fail in immunofluorescence.

For AMSH2 antibodies specifically, adherence to these standards is essential as studies have shown that many commercially available antibodies do not meet basic validation criteria .

What are the relative advantages of antibody-based versus genetic approaches for studying AMSH2 function?

When studying AMSH2 and the TGF-β pathway, researchers successfully combined genetic approaches (siRNA knockdown) with antibody methods and reporter assays to demonstrate AMSH2's role as a positive regulator of TGF-β signaling .

How does the choice of detection method affect the interpretation of AMSH2 antibody results?

Different detection methods can significantly impact AMSH2 antibody results interpretation:

• Western blot:

  • Advantages: Provides molecular weight information, can differentiate between specific and non-specific binding

  • Limitations: Denatures proteins, may lose conformational epitopes

  • Interpretation considerations: Band size verification is critical; multiple bands require careful validation to distinguish true signal from cross-reactivity

• Immunoprecipitation:

  • Advantages: Maintains protein-protein interactions, can enrich low-abundance proteins

  • Limitations: Success highly dependent on antibody affinity in native conditions

  • Interpretation considerations: Requires confirmation of pulled-down proteins by Western blot or mass spectrometry

• Immunofluorescence:

  • Advantages: Reveals subcellular localization, potential co-localization with interacting partners

  • Limitations: Fixation can alter epitope accessibility, autofluorescence can confound results

  • Interpretation considerations: Proper controls (including knockout cells) are essential; cellular context affects localization patterns

• ELISA/multiplexed assays:

  • Advantages: Quantitative, high-throughput capability

  • Limitations: May not detect conformational changes, limited to known targets

  • Interpretation considerations: Standard curves critical for accurate quantification; matrix effects can influence results

• Flow cytometry:

  • Advantages: Single-cell analysis, can correlate with other cellular parameters

  • Limitations: Limited to cell surface or permeabilized intracellular proteins

  • Interpretation considerations: Permeabilization protocol affects antibody accessibility; fluorophore choice impacts sensitivity

According to validation studies, success in one application does not predict success in another. Interestingly, success in immunofluorescence is the best predictor of performance in both Western blot and immunoprecipitation applications .

For optimal interpretation, method-specific controls should always be included, and findings should be confirmed using orthogonal approaches whenever possible.