ANKRD55 Antibody

What is ANKRD55 and what is its molecular structure?

ANKRD55, or Ankyrin repeat domain containing protein 55, is a 614 amino acid protein with a calculated molecular weight of 68 kDa, containing 9 ANK repeats . It belongs to the ankyrin family and may exist in three alternatively spliced isoforms . The protein is encoded by the ANKRD55 gene located on chromosome 5q11. ANKRD55 has been primarily detected in human samples, with current antibodies showing reactivity specifically to human ANKRD55 .

Recent structural analysis indicates that ANKRD55 proteins predominantly localize to the nuclei of cells, suggesting potential roles in gene regulation or nuclear processes . The ankyrin repeat domains typically facilitate protein-protein interactions, which may be central to ANKRD55's biological function.

What are the available ANKRD55 antibody specifications for research use?

Multiple validated ANKRD55 antibodies are available for research, with the following specifications:

The 24203-1-AP antibody is generated against an ANKRD55 fusion protein (Ag21271) and purified using antigen affinity methods . The PACO35926 antibody is derived using recombinant Human Ankyrin repeat domain-containing protein 55 (residues 151-326) as the immunogen and is purified using Protein G .

What experimental applications are supported by current ANKRD55 antibodies?

ANKRD55 antibodies have been validated for multiple experimental applications with specific recommended dilutions:

For IHC applications with 24203-1-AP, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative . Researchers are advised to titrate these antibodies in their specific testing systems to obtain optimal results as signal strength can be sample-dependent .

How should researchers optimize ANKRD55 antibody protocols for different tissue types?

When working with ANKRD55 antibodies across different tissue types, consider the following optimization strategies:

For immunohistochemistry applications, antigen retrieval conditions significantly impact staining quality. With human tissues, TE buffer at pH 9.0 is recommended as the primary approach, while citrate buffer at pH 6.0 serves as an alternative method . Different tissue fixation methods may require protocol adjustments - formalin-fixed paraffin-embedded tissues typically need more stringent antigen retrieval than frozen sections.

For Western blot detection, optimization should focus on:

• Lysate preparation methods (RIPA buffer with protease inhibitors is suitable for most applications)

• Loading concentration (25-50 μg total protein is recommended for detecting ANKRD55)

• Transfer conditions (wet transfer at 100V for 60-90 minutes)

• Blocking buffer composition (5% non-fat milk or BSA in TBST)

• Primary antibody incubation time (overnight at 4°C often yields better results than shorter incubations)

For immunofluorescence applications, specificity can be enhanced by:

• Using lower antibody concentrations (starting at 1:100 and titrating as needed)

• Extending incubation periods (12-16 hours at 4°C)

• Including appropriate controls (both negative controls and positive controls in cells known to express ANKRD55, such as HeLa cells)

What are the critical considerations when interpreting ANKRD55 antibody staining patterns?

When interpreting ANKRD55 staining patterns, researchers should consider:

Subcellular localization: ANKRD55 proteins primarily localize to the nuclei of cells , though isoform-specific localization patterns may vary. Any significant cytoplasmic staining should be carefully validated.

Cell type specificity: ANKRD55 expression is predominantly detected in CD4+ T cells, with minimal expression in CD8+, CD14+, CD19+, and CD56+ cells . This restricted expression pattern should be considered when evaluating staining in mixed cell populations.

Signal specificity verification:

• Include known positive controls (HeLa cells, L02 cells for Western blot)

• Perform peptide competition assays to confirm antibody specificity

• Compare results across multiple detection methods (WB, IHC, IF)

• Consider genetic approaches (siRNA knockdown) to validate antibody specificity

Genetic variant considerations: The rs6859219 variant affects ANKRD55 expression levels , with homozygotes for the risk allele producing more than four times the transcript copies compared to those with the protective allele. This genetic variation could impact staining intensity in patient-derived samples.

What is the established relationship between ANKRD55 and Multiple Sclerosis?

ANKRD55 has emerged as a significant gene in Multiple Sclerosis (MS) research. MS is characterized by demyelination and chronic neurodegenerative damage to the central nervous system, with autoimmune mechanisms playing a central role .

Research has established that:

• An intronic variant in ANKRD55, rs6859219, is a genetic risk factor for MS

• This variant functions as a cis-expression quantitative trait locus (eQTL), significantly affecting ANKRD55 transcript levels in PBMCs and CD4+ T cells

• ANKRD55 produces three different transcript variants (Ensembl isoforms 001, 005, and 007)

• The MS-associated genetic variant substantially increases the production of these transcripts

• This expression pattern is specific to CD4+ T cells, which are crucial for protective immune responses and thought to be dysregulated in MS

• Homozygotes for the risk allele produce more than four times more transcript copies than those with the protective allele

The nuclear localization of ANKRD55 proteins suggests they may play a role in transcriptional regulation or other nuclear processes relevant to MS pathogenesis . These findings collectively point to ANKRD55 as a key gene in the 5q11 chromosome region associated with immune response and MS susceptibility.

What methodologies are most effective for studying ANKRD55's role in neuroinflammatory conditions?

For investigating ANKRD55's role in neuroinflammatory conditions, researchers should employ a multi-faceted approach:

Transcript analysis techniques:

• Quantitative PCR (qPCR) using specific primer pairs that provide best coverage for ANKRD55 isoforms (as used in research with IDT, Cat. No. Hs.PT.58.27501603201)

• Normalization with appropriate housekeeping genes (ACTB and GAPDH have been successfully used)

• Digital PCR for absolute quantification of transcript variants

Protein detection and localization:

• Immunofluorescence microscopy to confirm nuclear localization in various cell types

• Cell fractionation followed by Western blot to quantify nuclear vs. cytoplasmic distribution

• Co-immunoprecipitation to identify interaction partners

• Chromatin immunoprecipitation if transcriptional regulatory functions are suspected

Functional studies:

• siRNA or CRISPR-based knockdown/knockout in relevant cell types (CD4+ T cells)

• Analysis of downstream transcriptional effects using RNA-Seq

• Assessment of T cell activation, proliferation, and cytokine production in ANKRD55-modified cells

• EAE (experimental autoimmune encephalomyelitis) models comparing wild-type and ANKRD55-modulated systems

Genetic association studies:

• Genotyping of rs6859219 and other ANKRD55 variants in MS cohorts

• Integration with clinical parameters to identify genotype-phenotype correlations

• eQTL analysis in relevant tissues and cell types

How does the interaction between ANKRD55 and IFT-B-like complex in microglia inform our understanding of its function?

Recent research has uncovered an interaction between ANKRD55 and an intraflagellar transport B (IFT-B)-like complex in microglia , providing novel insights into ANKRD55's functional role. This discovery suggests potential involvement in intracellular transport mechanisms that may be relevant to microglial function in neuroinflammation.

Methodologically, this interaction was identified through sophisticated protein-protein interaction studies:

• Protein interactome analysis using STRING database revealed associations between ANKRD55 and components of the IFT-B complex

• Mass spectrometry analysis using a modified spectral counting method (Normalized Spectral Abundance Factor, NSAF) helped quantify these interactions

• Proteomic data identified specific IFT components including IFT74, IFT46, and IFT56 (TTC26) as interaction partners

These findings suggest ANKRD55 may participate in:

• Microglial motility and surveillance functions

• Intracellular transport of proteins and organelles

• Signaling pathways relevant to neuroinflammation

• Potential cilia-related functions in neural cells

Researchers investigating this interaction should employ co-immunoprecipitation, proximity ligation assays, and fluorescence resonance energy transfer (FRET) techniques to further characterize the spatial and temporal dynamics of these interactions in microglia and other neural cell types.

What approaches should be used to resolve contradictory data regarding ANKRD55 expression patterns?

When encountering contradictory data regarding ANKRD55 expression patterns, researchers should implement the following systematic approach:

Technical validation:

• Compare antibody specifications and epitopes across studies

• Evaluate cell isolation techniques and purity of populations studied

• Assess RNA integrity in transcript analyses

• Consider fixation and permeabilization methods that might affect epitope accessibility

Biological considerations:

• Genetic background of samples (rs6859219 genotype significantly affects expression levels)

• Cell activation state (expression may be modulated by activation signals)

• Tissue/cell source variability (primary cells vs. cell lines)

• Splice variant detection (ensure methods can distinguish between the three identified transcript variants)

Resolution strategies:

• Multi-modal detection (combining RNA and protein measurements)

• Single-cell analysis to resolve heterogeneity within populations

• Temporal analysis to identify dynamic changes in expression

• Genotype-stratified analysis

• Use of multiple antibodies targeting different epitopes

For instance, while ANKRD55 transcripts were detected in CD4+ T cells but virtually absent in CD8+, CD14+, CD19+, and CD56+ cells , contradictory findings might emerge under different activation conditions or in different disease states. Similarly, the processed noncoding transcript 007 was the most highly expressed variant in CD4+ T cells but was not detected in Jurkat, U937, and SH-SY5Y cell lines , highlighting the importance of cell type considerations in expression analysis.

What are common pitfalls in ANKRD55 antibody applications and how can they be addressed?

Researchers working with ANKRD55 antibodies may encounter several technical challenges that can be systematically addressed:

Weak or absent signal in Western blots:

• Increase protein loading (up to 50-75 μg total protein)

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

• Use more sensitive detection methods (ECL Plus or SuperSignal West Femto)

• Verify sample preparation (ensure complete cell lysis and include protease inhibitors)

• Consider native vs. reducing conditions if protein conformation affects epitope accessibility

High background in immunostaining:

• Increase blocking time and concentration (5% BSA or normal serum for 1-2 hours)

• Reduce primary antibody concentration (begin with 1:100 and titrate to 1:200 or 1:500)

• Extend washing steps (5-6 washes of 5-10 minutes each)

• Use appropriate negative controls (isotype control and secondary-only controls)

• Pre-absorb antibody with cell/tissue lysate to reduce non-specific binding

Inconsistent results across experiments:

• Standardize sample collection and processing

• Aliquot antibodies to avoid freeze-thaw cycles

• Prepare fresh working solutions for each experiment

• Include positive controls in each experiment (HeLa or L02 cells)

• Maintain consistent incubation times and temperatures

Tissue-specific optimization considerations:

• For brain tissue: extended fixation time may require more rigorous antigen retrieval

• For blood cells: ensure proper permeabilization for nuclear antigens

• For formalin-fixed tissues: test multiple antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

How can researchers validate the specificity of ANKRD55 antibody detection in their experimental systems?

Rigorous validation of ANKRD55 antibody specificity is essential for reliable research outcomes. A comprehensive validation approach should include:

Controls and standards:

• Positive controls: Use cell lines with confirmed ANKRD55 expression (HeLa, L02)

• Negative controls: Include cell types with minimal expression (CD8+, CD14+, CD19+, CD56+ cells)

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

• Isotype controls: Use matched isotype antibodies to assess non-specific binding

Genetic validation approaches:

• siRNA or shRNA knockdown: Compare staining in cells with reduced ANKRD55 expression

• Overexpression systems: Analyze cells transfected with ANKRD55 expression constructs

• CRISPR/Cas9 knockout: Generate complete knockout cells as definitive negative controls

Multi-method confirmation:

• Compare results across different detection methods (WB, IHC, IF, ELISA)

• Use antibodies targeting different epitopes of ANKRD55

• Correlate protein detection with mRNA expression data

• Consider mass spectrometry validation for definitive protein identification

Isoform-specific considerations:

• Determine which isoforms (001, 005, 007) are detected by the antibody

• Use PCR primers specific to each isoform to correlate transcript presence with protein detection

• Consider that the noncoding transcript 007 was the most highly expressed variant in CD4+ T cells but absent in several cell lines