ANKRD42 Antibody

What is ANKRD42 protein and what is its function in cellular biology?

ANKRD42 (Ankyrin repeat domain-containing protein 42) is a human protein encoded by the ANKRD42 gene. It is also known by alternative names including SARP and PPP1R79. The protein consists of 389 amino acids with a molecular weight of approximately 43kDa . While much remains to be elucidated about its specific function, ANKRD42 has been reported to interact with proteins including PPP1CC (Protein Phosphatase 1 Catalytic Subunit Gamma) and UBC (Ubiquitin C), suggesting potential roles in phosphorylation-dependent signaling and ubiquitin-mediated processes . The ankyrin repeat domains typically mediate protein-protein interactions, indicating ANKRD42 may function as a scaffolding or adaptor protein in cellular signaling networks. Current research suggests this protein may have regulatory functions, but detailed characterization requires further investigation.

What types of ANKRD42 antibodies are available for research applications?

Several types of ANKRD42 antibodies are currently available for research purposes, with the most common being rabbit polyclonal antibodies targeting the N-terminal region of human ANKRD42 . These antibodies are typically affinity-purified and supplied in unconjugated form, though custom conjugation services may be available through some suppliers . The antibodies are validated for various applications including Western blotting/immunoblotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) . Most commercially available antibodies have been raised against synthetic peptides derived from the N-terminal sequence of the human protein, though the exact epitope sequence varies between products .

What are the optimal conditions for using ANKRD42 antibodies in Western blotting?

For optimal Western blotting results with ANKRD42 antibodies, researchers should consider the following methodological approach:

• Sample preparation: Use RIPA or NP-40 based lysis buffers with protease inhibitors to extract total protein from tissues or cells known to express ANKRD42.

• Protein separation: Load 20-50μg of total protein per lane on 10-12% SDS-PAGE gels, as the target protein has a molecular weight of approximately 43kDa .

• Transfer: Use PVDF membranes for protein transfer (nitrocellulose is also acceptable but may provide lower sensitivity).

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

• Primary antibody incubation: Dilute the ANKRD42 antibody to working concentration (typically 1:500-1:2000 for most commercial antibodies) . Incubate overnight at 4°C with gentle agitation.

• Secondary antibody selection: Use appropriate HRP-conjugated anti-rabbit IgG secondary antibodies, as most ANKRD42 antibodies are rabbit-derived .

• Detection: Standard ECL detection methods are suitable for visualizing the target band.

Researchers should be aware that post-translational modifications may affect the observed molecular weight, and validation with positive controls and blocking peptides is recommended to confirm specificity .

How should ANKRD42 antibodies be prepared and stored to maintain optimal activity?

To maintain optimal antibody activity, researchers should follow these storage and handling recommendations:

• Short-term storage (up to several weeks): Store antibodies at 2-8°C in their original formulation, which typically includes preservatives such as sodium azide (0.02-0.09%) .

• Long-term storage: For extended periods, aliquot antibodies into small volumes (10-20μl) to avoid repeated freeze-thaw cycles and store at -20°C or below .

• Thawing: When needed, thaw aliquots at room temperature or at 4°C, and briefly centrifuge to collect the solution at the bottom of the tube.

• Dilution: Dilute antibodies only immediately prior to use in appropriate buffer systems. Most ANKRD42 antibodies are supplied in PBS (pH 7.4) with sodium azide and may contain stabilizers like sucrose (2%) .

• Working dilutions: Once diluted to working concentration, use within 24 hours for optimal results.

• Avoid repeated freeze-thaw cycles: Multiple freeze-thaw cycles can lead to denaturation and loss of antibody activity. Limit to 3-5 cycles maximum.

Most commercial ANKRD42 antibodies have a shelf life of approximately one year from the date of receipt when stored properly . Always consult the manufacturer's specific recommendations as formulations can vary between suppliers.

What controls should be included when validating ANKRD42 antibodies for a new experimental system?

A comprehensive validation strategy for ANKRD42 antibodies should include the following controls:

• Positive tissue/cell controls: Include samples known to express ANKRD42 based on tissue expression databases. While comprehensive expression data is limited, researchers should consider tissues where ankyrin repeat proteins are typically found.

• Negative controls: Include samples where ANKRD42 expression is expected to be absent or use siRNA/shRNA knockdown samples if available.

• Blocking peptide competition: Use the immunogenic peptide (available as catalog # AAP41193 for some antibodies) to pre-absorb the antibody and confirm signal specificity. A significant reduction in signal intensity should be observed in blocked samples.

• Loading controls: Include appropriate housekeeping proteins (β-actin, GAPDH, etc.) to normalize protein loading and enable quantitative comparisons.

• Molecular weight markers: Confirm that the detected band corresponds to the expected molecular weight of ANKRD42 (approximately 43kDa) .

• Secondary antibody-only control: Omit primary antibody to identify potential non-specific binding of the secondary antibody.

• Isotype control: Use a non-specific rabbit IgG at the same concentration to identify potential non-specific binding.

This systematic approach to validation ensures that observed signals are genuine and specific to ANKRD42, particularly important when working with a relatively understudied protein.

What are common issues encountered when using ANKRD42 antibodies in immunohistochemistry and how can they be addressed?

When using ANKRD42 antibodies for immunohistochemistry, researchers may encounter several challenges that can be addressed with specific troubleshooting approaches:

Issue: Weak or absent signal

• Solution: Optimize antibody concentration (try 1:200-1:500 dilution range)

• Solution: Extend primary antibody incubation (overnight at 4°C)

• Solution: Enhance antigen retrieval by testing different methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0, with varied heating times)

• Solution: Use signal amplification systems (HRP-polymer or avidin-biotin complex)

Issue: High background staining

• Solution: Increase blocking duration (2-3 hours with 5-10% normal serum)

• Solution: Include 0.1-0.3% Triton X-100 in blocking solution to reduce non-specific binding

• Solution: Reduce primary antibody concentration

• Solution: Increase washing steps duration and frequency

Issue: Non-specific staining patterns

• Solution: Validate antibody specificity using blocking peptide competition

• Solution: Include tissue sections known to be negative for ANKRD42

• Solution: Use RNAi knockdown controls if available

Issue: Inconsistent staining across tissue sections

• Solution: Standardize fixation protocols (preferably 10% neutral buffered formalin for 24 hours)

• Solution: Ensure consistent section thickness (4-6μm recommended)

• Solution: Process all experimental and control sections simultaneously

Researchers should note that for immunohistochemistry applications, a working dilution range of 1:200-1:500 has been recommended for some commercial ANKRD42 antibodies , though optimization for specific tissue types is advisable.

How can ANKRD42 antibody specificity be verified when working with novel cell types or tissues?

Verifying ANKRD42 antibody specificity in novel experimental systems requires a multi-faceted approach:

• Western blot analysis: First perform Western blotting to confirm a single band at the expected molecular weight (43kDa) . Multiple bands may indicate cross-reactivity or post-translational modifications.

• Immunoprecipitation followed by mass spectrometry: Immunoprecipitate the target protein and analyze by mass spectrometry to confirm identity.

• Genetic approaches: Use CRISPR/Cas9 or siRNA to knock down ANKRD42 expression and demonstrate corresponding reduction in antibody signal.

• Heterologous expression systems: Express tagged ANKRD42 in a cell line with low endogenous expression and confirm co-localization of antibody signal with the tag.

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of ANKRD42 and compare the patterns.

• Blocking peptide competition: Pre-incubate the antibody with excess immunogenic peptide and demonstrate elimination or reduction of signal in all applications.

• RNA-protein correlation: Correlate protein detection with mRNA levels using RT-qPCR or RNA-seq data.

A comprehensive validation strategy combining several of these approaches provides robust evidence for antibody specificity, particularly important for relatively uncharacterized proteins like ANKRD42.

What quantification methods are most appropriate for ANKRD42 expression analysis in different experimental contexts?

Appropriate quantification methods for ANKRD42 expression analysis vary by experimental technique:

For Western blot analysis:

• Densitometric analysis using software like ImageJ, normalizing to loading controls (β-actin, GAPDH)

• Calculate relative expression using the formula:
  Relative Expression = (ANKRD42 band intensity / Loading control intensity) × 100

For immunohistochemistry/immunofluorescence:

• H-score method: H-score = Σ(i × Pi), where i = intensity (0-3) and Pi = percentage of cells (0-100%)

• Automated image analysis using software like QuPath or ImageJ with tissue classifier plugins

• Mean fluorescence intensity measurements for immunofluorescence

For ELISA-based quantification:

• Absolute quantification using standard curves with recombinant ANKRD42 protein

• The detection range for commercial ANKRD42 ELISA kits is typically 0.5-10ng/mL with a sensitivity of 0.1ng/mL

For qPCR correlation:
Integrate protein quantification with mRNA expression data using the 2^(-ΔΔCT) method to assess concordance between transcription and translation

When analyzing ANKRD42 expression data, researchers should consider:

• Biological replicates (minimum n=3) for statistical validity

• Technical replicates to assess methodological variability

• Appropriate statistical tests based on data distribution (parametric vs. non-parametric)

• Presentation of raw data alongside normalized values when possible

How can ANKRD42 antibodies be utilized in protein-protein interaction studies to elucidate its functional role?

Given the limited knowledge about ANKRD42 function, antibodies can be instrumental in uncovering its protein interaction network through these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use ANKRD42 antibodies to pull down the protein complex from cell lysates, followed by mass spectrometry analysis to identify interacting partners. Previous studies suggest PPP1CC and UBC as potential interactors .

Co-IP Protocol Outline:

• Lyse cells in non-denaturing buffer (e.g., 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, protease inhibitors)

• Pre-clear lysate with Protein A/G beads

• Incubate cleared lysate with 2-5μg ANKRD42 antibody overnight at 4°C

• Add Protein A/G beads and incubate 2-4 hours

• Wash extensively and elute in SDS sample buffer

• Analyze by SDS-PAGE followed by immunoblotting or mass spectrometry

• Proximity Ligation Assay (PLA): Visualize protein-protein interactions in situ using ANKRD42 antibody in combination with antibodies against suspected interacting partners.

• ChIP-seq integration: If ANKRD42 functions in transcriptional regulation, combine ChIP-seq data with co-IP to identify DNA-protein complexes.

• Bimolecular Fluorescence Complementation (BiFC): Though this requires cloning rather than antibodies directly, results can be validated using ANKRD42 antibodies.

• FRET/FLIM analysis: Use fluorescently labeled antibodies to detect protein proximities through fluorescence resonance energy transfer.

Based on the predicted functions of ankyrin repeat domains and the known interactions with PPP1CC (a phosphatase subunit), researchers should particularly explore ANKRD42's potential role in phosphorylation-dependent signaling pathways.

What approaches can be used to study ANKRD42 post-translational modifications using available antibodies?

To investigate post-translational modifications (PTMs) of ANKRD42, researchers can employ several antibody-dependent strategies:

• Immunoprecipitation followed by PTM-specific detection:

  • Immunoprecipitate ANKRD42 using available antibodies

  • Probe with antibodies specific to PTMs (phospho-, ubiquitin-, SUMO-, etc.)

  • Alternatively, analyze by mass spectrometry for comprehensive PTM mapping

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Detect ANKRD42 using the antibody to identify charge variants indicating PTMs

  • Compare patterns before and after phosphatase treatment

• Phosphorylation-specific analysis:

  • Treatment with phosphatase inhibitors versus phosphatases

  • Mobility shift assays in Phos-tag™ gels

  • Given the interaction with PPP1CC (a phosphatase) , phosphorylation may be particularly relevant

• Ubiquitination studies:

  • Given the reported interaction with UBC , investigate ubiquitination using:

  • Proteasome inhibitors (MG132) to accumulate ubiquitinated proteins

  • Immunoprecipitation under denaturing conditions followed by ubiquitin detection

• Stimulus-dependent modifications:

  • Treat cells with various stimuli (growth factors, stressors)

  • Immunoprecipitate ANKRD42 and analyze PTM changes

• Correlation with function:

  • Engineer PTM site mutants and assess functional consequences

  • Validate findings using the antibodies to detect wild-type protein

Current commercial antibodies target the N-terminal region of ANKRD42 , so researchers should consider that PTMs in this region might affect antibody binding and potentially yield false-negative results.

How can ANKRD42 antibodies be integrated into multi-omics research approaches to understand its biological context?

Integrating ANKRD42 antibodies into multi-omics research frameworks can provide comprehensive insights into its biological function and relevance:

• Proteomics integration:

  • Use ANKRD42 antibodies for immunoprecipitation followed by mass spectrometry

  • Create protein interaction networks incorporating both direct (co-IP) and indirect (proximity labeling) interactions

  • Quantify ANKRD42 expression across tissue/cell types and correlate with proteome-wide changes

• Transcriptomics correlation:

  • Compare protein expression patterns (detected via antibodies) with mRNA expression

  • Identify discordance that might indicate post-transcriptional regulation

  • Document co-expression patterns to identify functionally related gene clusters

• Functional genomics validation:

  • Validate CRISPR/RNAi screens with antibody-based detection methods

  • Assess consequences of genetic manipulation on ANKRD42 protein levels and localization

  • Use immunofluorescence with ANKRD42 antibodies to determine subcellular localization changes in genetic perturbation models

• Phosphoproteomics integration:

  • Given ANKRD42's interaction with PPP1CC , correlate phosphoproteome changes with ANKRD42 expression/depletion

  • Identify phosphosites on ANKRD42 and generate phospho-specific antibodies if not commercially available

• Spatial transcriptomics and proteomics correlation:

  • Use immunohistochemistry with ANKRD42 antibodies to map protein distribution

  • Correlate with spatial transcriptomics data to identify regions of post-transcriptional regulation

• Clinical sample analysis:

  • Apply validated antibodies to tissue microarrays

  • Correlate expression patterns with clinical parameters and multi-omics datasets

For immunohistochemistry or immunofluorescence applications, most commercial ANKRD42 antibodies are recommended to be used at dilutions of 1:200-1:500 , though optimization based on specific tissue types and fixation methods is advisable.

What are the best approaches for multiplexed detection systems incorporating ANKRD42 antibodies?

For researchers interested in simultaneous detection of ANKRD42 with other proteins, several multiplexed detection strategies can be employed:

• Multiplexed immunofluorescence:

  • Use ANKRD42 rabbit polyclonal antibodies in combination with antibodies from different host species

  • Select fluorophores with minimal spectral overlap

  • Consider sequential staining for antibodies from the same species

  • Employ Tyramide Signal Amplification (TSA) to allow multiple antibodies from the same species

• Mass cytometry (CyTOF):

  • Conjugate ANKRD42 antibodies with rare earth metals

  • Combine with dozens of other metal-tagged antibodies

  • Particularly useful for single-cell analysis in heterogeneous samples

• Proximity Ligation Assay (PLA) multiplexing:

  • Combine ANKRD42 antibodies with antibodies against potential interacting partners

  • Use different fluorophore-labeled PLA probes to visualize multiple interactions simultaneously

• Multiplex immunohistochemistry:

  • Sequential staining with ANKRD42 antibodies and other targets

  • Between rounds, strip or quench previous detection systems

  • Alternative: spectral unmixing of chromogenic dyes

• Microfluidic-based multiplexed immunoassays:

  • Spatially separate capture antibodies including anti-ANKRD42

  • Detect multiple proteins simultaneously from small sample volumes

• Imaging Mass Cytometry (IMC):

  • Apply metal-conjugated ANKRD42 antibodies to tissue sections

  • Laser ablation coupled with mass spectrometry enables detection of 40+ proteins

  • Provides spatial context at subcellular resolution

When designing multiplexed systems, consider that most ANKRD42 antibodies are rabbit-derived polyclonals , which may limit compatibility with other rabbit antibodies unless specialized multiplexing techniques are employed. The unconjugated format of commercially available ANKRD42 antibodies allows researchers flexibility in selecting appropriate secondary detection systems for their specific multiplexing approach.

How might ANKRD42 antibodies contribute to understanding its potential role in specific cellular pathways?

Given ANKRD42's protein interactions with PPP1CC (a phosphatase catalytic subunit) and UBC (ubiquitin C) , antibodies can be leveraged to explore its potential roles in several cellular pathways:

• Phosphorylation-dependent signaling:

  • Use ANKRD42 antibodies in co-localization studies with phosphatases and kinases

  • Perform phosphatase activity assays after ANKRD42 immunodepletion

  • Investigate phosphoproteomic changes after ANKRD42 knockdown/overexpression

• Ubiquitin-proteasome system:

  • Explore ANKRD42's potential adapter role in protein degradation pathways

  • Use antibodies to track ANKRD42 localization during proteasomal stress

  • Investigate co-localization with ubiquitinated proteins after proteasome inhibition

• Subcellular dynamics:

  • Track ANKRD42 localization during cell cycle progression using immunofluorescence

  • Investigate potential translocation under various cellular stresses

  • Examine co-localization with organelle markers under different conditions

• Protein complex assembly:

  • Use antibodies in size-exclusion chromatography followed by immunoblotting

  • Identify different ANKRD42-containing complexes under various cellular conditions

  • Perform blue native PAGE followed by immunodetection

• Transcriptional regulation:

  • Investigate potential nuclear localization using fractionation and immunodetection

  • Perform ChIP assays if nuclear localization is observed

  • Correlate with transcriptomic changes in ANKRD42-depleted cells

For immunohistochemistry or immunofluorescence applications, researchers should optimize the working dilution within the recommended range (typically 1:200-1:500 for commercial antibodies) and verify specificity in their experimental system.

What technical considerations should be addressed when developing custom ANKRD42 antibodies for specialized research applications?

Researchers considering the development of custom ANKRD42 antibodies should address these technical considerations:

• Epitope selection strategy:

  • Avoid the N-terminal region already targeted by commercial antibodies if seeking complementary reagents

  • Consider the protein's domain structure – ankyrin repeat domains may offer conserved epitopes

  • Use epitope prediction algorithms to identify antigenic regions with high surface probability

  • Assess sequence conservation across species if cross-reactivity is desired

• Immunization approach:

  • For phospho-specific antibodies, design phosphopeptides based on predicted phosphorylation sites

  • For domain-specific antibodies, consider recombinant protein fragments rather than peptides

  • Design fusion proteins to enhance immunogenicity of poorly antigenic regions

• Validation requirements:

  • Include knockout/knockdown controls in validation plans

  • Pre-arrange access to tissues from multiple species if cross-reactivity is important

  • Plan for extensive cross-validation with existing commercial antibodies

  • Design competing peptides for specificity testing

• Special modifications for advanced applications:

  • For super-resolution microscopy: consider smaller antibody formats (Fab, nanobodies)

  • For live-cell imaging: plan for internalization sequences or cell-penetrating peptides

  • For multiplexing: design conjugation strategies compatible with other antibodies

• Production considerations:

  • For monoclonal development, plan for extensive screening to identify clones recognizing native protein

  • For recombinant antibodies, design constructs allowing site-specific conjugation

  • Consider humanization if therapeutic applications might be explored later

Custom antibody development should be guided by the specific research questions being addressed and the limitations identified in commercially available antibodies, such as the predominance of N-terminal targeting in current products .

How can researchers interpret contradictory results when using different ANKRD42 antibodies in the same experimental system?

When facing contradictory results with different ANKRD42 antibodies, researchers should implement a systematic troubleshooting approach:

• Epitope mapping comparison:

  • Determine the exact epitopes recognized by each antibody

  • Commercial ANKRD42 antibodies typically target the N-terminal region but may recognize different sequences

  • Consider potential epitope masking by protein interactions or post-translational modifications

• Validation status assessment:

  • Review validation data for each antibody (Western blot, immunoprecipitation, peptide competition)

  • Consider performing additional validation if documentation is limited

  • Verify each antibody against positive and negative controls

• Methodological differences analysis:

  • Compare protocols used with each antibody (fixation methods, antigen retrieval, blocking conditions)

  • Standardize experimental conditions and test all antibodies under identical parameters

  • Assess buffer compatibility with each antibody's optimal working conditions

• Biological interpretation framework:

  • Consider that different antibodies might detect different isoforms or post-translationally modified variants

  • Explore if contradictions reflect biologically relevant phenomena (e.g., conformation changes, complex formation)

  • Use orthogonal techniques (mass spectrometry, genetic approaches) to resolve contradictions

• Technical resolution approach:

  • Create a decision matrix comparing antibody performance across multiple validation criteria

  • Weight evidence based on validation robustness

  • Consider developing consensus protocols that work reasonably well with multiple antibodies

• Documentation and reporting:

  • Thoroughly document all antibody characteristics (catalog numbers, lots, dilutions)

  • Report contradictory findings transparently in publications

  • Consider contributing validation data to antibody validation repositories

For truly contradictory results, researchers should consider that ANKRD42 remains relatively uncharacterized, and discrepancies might provide valuable insights into protein behavior under different conditions or in different conformational states.

What methodological approaches can optimize ANKRD42 antibody use in human tissue microarrays for disease correlation studies?

To optimize ANKRD42 antibody applications in tissue microarray (TMA) studies for disease correlations, researchers should implement these methodological approaches:

• Antibody validation for TMA application:

  • Validate antibody on whole tissue sections before TMA application

  • Include positive and negative control cores in every TMA

  • Verify specificity using peptide competition on representative cores

• Optimization protocol for immunohistochemistry:

  • Titrate antibody concentration precisely (starting with recommended 1:200-1:500 dilution)

  • Test multiple antigen retrieval methods (pH 6.0 citrate and pH 9.0 EDTA buffers)

  • Optimize detection systems (polymer-HRP vs. ABC method)

  • Standardize development times using timed protocols

• Scoring system development:

  • Establish clear scoring criteria (H-score, Allred score, or percentage positive cells)

  • Train multiple pathologists for consistent interpretation

  • Consider automated image analysis for objectivity

  • Document both intensity and distribution patterns

• Quality control measures:

  • Include serial sections stained with isotype controls

  • Apply tissue orientation markers for accurate core identification

  • Include multiple cores per case to account for heterogeneity

  • Process all TMAs in a single batch when possible

• Correlation analysis design:

  • Collect comprehensive clinicopathological data for all cases

  • Design statistical analysis plan before staining

  • Plan for multivariate analysis to identify confounding factors

  • Calculate required sample sizes for adequate statistical power

• Technology integration:

  • Consider multiplexed approaches to correlate ANKRD42 with other markers

  • Integrate with genomic data from the same cohort when available

  • Document subcellular localization patterns that may have functional significance