AHNAK2 Antibody

What is AHNAK2 and why is it significant in research?

AHNAK2 (AHNAK nucleoprotein 2) is a large ubiquitously expressed protein with a molecular mass of approximately 617 kDa. It is also known by alternative names including C14orf78, KIAA2019, and Protein AHNAK2 . Research interest in AHNAK2 has grown due to its emerging roles in disease pathways, particularly in cancer. Recent studies indicate that AHNAK2 promotes pancreatic ductal adenocarcinoma progression by stabilizing c-MET . Additionally, AHNAK has been implicated in increased IL-6 production in CD4+ T cells and may serve as a potential diagnostic biomarker for recurrent pregnancy loss . These findings highlight AHNAK2 as a promising target for both fundamental research and potential therapeutic development in multiple disease contexts.

What are the key structural and functional characteristics of AHNAK2?

AHNAK2 is characterized by its exceptionally large size (617 kDa) which presents unique challenges for experimental detection and analysis . The protein is encoded by the AHNAK2 gene (Gene ID: 113146), with a GenBank accession number of BC049216 . While complete structural characterization remains ongoing, functional studies have begun to elucidate its biological roles, particularly in disease contexts such as cancer progression. Its ability to maintain c-MET stability suggests AHNAK2 may function as a scaffold protein involved in receptor tyrosine kinase signaling pathways critical for cell growth and survival . When designing experiments to study AHNAK2, researchers should consider its large molecular weight, which often requires special protocols for detection, particularly in Western blot applications.

What cell types and tissues express AHNAK2?

Based on validated experimental data, AHNAK2 expression has been confirmed in multiple human cell lines and tissues:

This widespread expression pattern suggests AHNAK2 may have diverse functions depending on cellular context. When designing experiments, these validated systems can serve as positive controls for AHNAK2 detection .

What are the optimal detection methods for AHNAK2 and their respective protocols?

Multiple experimental approaches can be used to detect and study AHNAK2, each with specific advantages and recommended protocols:

For IHC applications, antigen retrieval is typically performed using TE buffer (pH 9.0) or alternatively with citrate buffer (pH 6.0) . For optimal results, researchers should titrate antibodies for their specific experimental system and include appropriate positive controls from the validated cell types or tissues listed above.

What special considerations should be made when detecting AHNAK2 via Western blot?

AHNAK2's exceptionally large molecular weight (617 kDa) presents unique challenges for Western blot detection . To optimize results:

• Use low percentage (3-5%) polyacrylamide gels or gradient gels to allow proper resolution of high molecular weight proteins

• Implement extended transfer times (often overnight at low voltage) to ensure complete transfer

• Consider adding SDS (0.1%) to the transfer buffer to facilitate migration of the large protein

• Employ stringent lysis conditions to ensure complete protein solubilization

• Include protease inhibitors to prevent degradation during sample preparation

• Start with the recommended antibody dilution range (1:500-1:2000) and optimize as needed

• Use high molecular weight proteins as loading controls rather than standard lower molecular weight controls

Following these specialized protocols will significantly improve the likelihood of successful AHNAK2 detection by Western blot.

How should immunohistochemistry protocols be optimized for AHNAK2 detection?

For optimal AHNAK2 detection in tissue sections, researchers should follow these IHC protocol modifications:

• Antigen retrieval: Use TE buffer at pH 9.0 as the preferred method; citrate buffer at pH 6.0 can serve as an alternative

• Antibody dilution: Begin with a dilution range of 1:50-1:500 for paraffin-embedded tissues

• Positive controls: Include human heart, skin, renal cell carcinoma, or pancreatic cancer tissues as validated positive controls

• Blocking: Use sufficient blocking steps to minimize background, particularly important when working with human tissues

• Incubation time: Consider extended primary antibody incubation (overnight at 4°C) to improve signal strength

• Detection system: Use high-sensitivity detection systems compatible with rabbit primary antibodies

For example, successful staining has been demonstrated in paraffin-embedded human skin tissue using ab224061 at 1/200 dilution and in human heart tissue using appropriate retrieval methods .

How can researchers troubleshoot weak or absent AHNAK2 signal in Western blot applications?

When encountering difficulties detecting AHNAK2 by Western blot, consider these troubleshooting approaches:

Remember that the calculated molecular weight of AHNAK2 is 617 kDa , which is beyond the range of many standard protein ladders. Using HeLa or HEK-293 cell lysates as positive controls can help establish the correct band position .

What are common pitfalls in AHNAK2 immunostaining and how can they be addressed?

Researchers may encounter several challenges when performing IHC or IF for AHNAK2 detection:

• High background staining:

  • Increase blocking time and concentration (5% BSA or normal serum)

  • Optimize antibody dilution (start with 1:200 and adjust as needed)

  • Include additional washing steps with increased duration

  • Consider using more specific secondary antibodies

• Weak or variable staining:

  • Optimize antigen retrieval (compare TE buffer pH 9.0 vs citrate buffer pH 6.0)

  • Test longer primary antibody incubation periods

  • Ensure tissue fixation and processing is optimal and consistent

  • Use amplification systems for signal enhancement

• Non-specific staining:

  • Validate antibody specificity using genetic approaches (knockdown/knockout)

  • Perform peptide competition assays to confirm specificity

  • Include appropriate negative controls (isotype controls, secondary-only controls)

Successful staining has been reported in multiple tissues including human skin (1/200 dilution) and human heart tissue with appropriate retrieval methods .

How can antibody specificity for AHNAK2 be rigorously validated?

Rigorous validation of AHNAK2 antibody specificity is essential for reliable research outcomes:

• Genetic validation:

  • CRISPR/Cas9 knockout of AHNAK2 in model cell lines

  • siRNA/shRNA-mediated knockdown with multiple constructs

  • Compare staining patterns between wildtype and AHNAK2-depleted samples

• Technical validation:

  • Use multiple antibodies targeting different AHNAK2 epitopes

  • Pre-absorption tests with immunizing peptides

  • Western blot confirmation of specific detection at the correct molecular weight (617 kDa)

• Cross-reactivity assessment:

  • Test antibodies in tissues/cells from multiple species if cross-reactivity is claimed

  • Examine potential cross-reactivity with related proteins (e.g., AHNAK1)

  • Compare antibody staining patterns with mRNA expression data

• Documentation:

  • Record all validation experiments with appropriate controls

  • Report validation methods in publications

  • Consider antibodies with validation data in multiple applications (e.g., 17682-1-AP has validation in WB, IHC, and IF)

How can AHNAK2 be studied in the context of cancer progression mechanisms?

Recent research indicates AHNAK2 promotes pancreatic ductal adenocarcinoma by maintaining c-MET stability . To further investigate this and other cancer-related functions:

• Protein interaction studies:

  • Co-immunoprecipitation using validated AHNAK2 antibodies to identify binding partners

  • Proximity labeling approaches (BioID, APEX) to identify the AHNAK2 interactome

  • Mapping of interaction domains through truncation constructs

• Functional assessments:

  • CRISPR/Cas9 knockout or knockdown of AHNAK2 in cancer cell lines

  • Rescue experiments with wild-type vs. mutant AHNAK2

  • Phenotypic assays for migration, invasion, and proliferation

• Signaling pathway analysis:

  • Phospho-specific antibodies to assess downstream signaling

  • RNA-seq to identify transcriptional changes following AHNAK2 manipulation

  • Phospho-proteomics to identify global pathway alterations

• Clinical correlation:

  • IHC analysis of AHNAK2 expression in tumor microarrays

  • Correlation with patient outcomes and clinicopathological features

  • Multi-marker analysis with other cancer-related proteins

These approaches can help elucidate AHNAK2's role in cancer progression and potentially identify therapeutic opportunities.

What methods are recommended for studying AHNAK2 in immune regulation?

To investigate AHNAK2's reported role in immune regulation, particularly regarding IL-6 production in CD4+ T cells , consider these methodological approaches:

• Expression profiling:

  • Flow cytometry with validated AHNAK2 antibodies to quantify expression in immune cell subsets

  • Single-cell RNA-seq to identify expression patterns across immune populations

  • Correlation of AHNAK2 expression with immune cell activation states

• Functional studies:

  • CRISPR/Cas9 or siRNA-mediated AHNAK2 depletion in primary immune cells or relevant cell lines

  • Cytokine profiling (ELISA, Luminex) following AHNAK2 manipulation

  • Chromatin immunoprecipitation (ChIP) to assess transcriptional regulation of immune genes

• In vivo models:

  • Conditional AHNAK2 knockout in specific immune cell populations

  • Immune challenge models to assess functional consequences

  • Adoptive transfer experiments to determine cell-intrinsic effects

• Translational approaches:

  • Analysis of AHNAK2 expression in patient-derived immune cells across disease states

  • Correlation with inflammatory biomarkers

  • Ex vivo manipulation of patient samples

These approaches can help define AHNAK2's role in immune regulation and potential relevance to inflammatory or autoimmune conditions.

How can researchers investigate post-translational modifications of AHNAK2?

As a large protein, AHNAK2 likely undergoes various post-translational modifications (PTMs) that may regulate its function. To study these:

• Identification of modifications:

  • Immunoprecipitation with validated AHNAK2 antibodies followed by mass spectrometry

  • Phospho-specific antibodies to detect specific modification sites

  • In silico prediction of potential modification sites

• Functional analysis:

  • Site-directed mutagenesis of predicted modification sites

  • Comparison of wildtype vs. modification-deficient AHNAK2 in functional assays

  • Assessment of subcellular localization changes following stimulus-induced modifications

• Regulatory mechanisms:

  • Identification of kinases/enzymes responsible for AHNAK2 modifications

  • Analysis of modification dynamics under different cellular conditions

  • Inhibitor studies to block specific modifications

• Disease relevance:

  • Comparison of AHNAK2 modification patterns between normal and diseased tissues

  • Correlation of modification status with disease progression or therapeutic response

Understanding AHNAK2's post-translational modifications could provide insights into its regulation and identify potential points for therapeutic intervention.

How should researchers integrate and compare AHNAK2 data across different detection platforms?

When analyzing AHNAK2 expression or function using multiple methodologies, consider these integration approaches:

• Cross-platform normalization:

  • Establish reliable normalization standards for each platform

  • Include common positive controls (e.g., HeLa cells) across experiments

  • Use relative quantification rather than absolute values when comparing methods

• Discrepancy resolution:

  • When contradictory results emerge, first evaluate technical variables

  • Consider epitope availability in different sample preparations

  • Assess potential isoform or modification-specific detection

• Integrated analysis workflow:

  • Begin with most reliable detection method for your sample type

  • Confirm key findings with orthogonal approaches

  • Use spatial information from IHC/IF to contextualize quantitative data from WB or ELISA

• Reporting standards:

  • Clearly document all methodological details for each platform

  • Acknowledge limitations of each approach

  • Present both raw and normalized data when possible

What statistical approaches are appropriate for analyzing AHNAK2 expression in research samples?

When quantifying AHNAK2 expression across experimental conditions or sample types:

• Appropriate controls and normalization:

  • For Western blots: normalize to total protein rather than single housekeeping genes due to AHNAK2's large size

  • For IHC: use digital image analysis with internal control normalization

  • For qPCR: validate reference genes specifically in your experimental system

• Statistical methods:

  • For normally distributed data: parametric tests (t-test, ANOVA with appropriate post-hoc tests)

  • For non-parametric distributions: Mann-Whitney, Kruskal-Wallis tests

  • For grouped or paired samples: repeated measures approaches

  • For multiple variable analysis: multivariate analysis, principal component analysis

• Sample size considerations:

  • Perform power analysis to determine adequate sample size

  • Report biological and technical replicates separately

  • Consider hierarchical statistical approaches for nested experimental designs

• Visualization approaches:

  • Box plots to show distribution characteristics

  • Scatter plots with mean/median indicators to show individual data points

  • For IHC quantification: consider H-score or Allred scoring systems

These statistical best practices will ensure robust analysis of AHNAK2 expression data in research applications.

How can contradictory findings about AHNAK2 function be reconciled in the literature?

When faced with conflicting reports about AHNAK2 biology, researchers should systematically evaluate:

• Methodological differences:

  • Antibody epitope specificity: Different antibodies may detect distinct regions of this large protein

  • Detection techniques: Sensitivity and specificity vary across methods

  • Sample preparation: Different lysis or fixation protocols may affect epitope availability

• Biological variables:

  • Cell/tissue-specific functions: AHNAK2 may have context-dependent roles

  • Experimental conditions: Growth conditions, cell density, and other variables may influence AHNAK2 function

  • Genetic background: Consider strain, species, or donor differences

• Reconciliation strategies:

  • Design experiments specifically addressing contradictions

  • Use multiple validated antibodies targeting different regions

  • Implement both genetic and pharmacological approaches

  • Consider temporal dynamics of AHNAK2 expression/function

• Collaborative approach:

  • Engage with authors of conflicting studies

  • Consider reproducibility initiatives with standardized protocols

  • Share detailed methodological information beyond standard publication requirements

By systematically addressing contradictions, researchers can advance understanding of context-dependent AHNAK2 biology rather than merely identifying "correct" versus "incorrect" findings.