ANKRD1 Antibody

What is ANKRD1 and what cellular functions does it perform?

ANKRD1 belongs to the conserved muscle ankyrin repeat protein (MARP) family, whose expression is induced in response to physiologic stress, injury, and hypertrophy. It functions primarily as a nuclear transcription repressor that regulates cardiac gene expression. ANKRD1 is mainly expressed in activated vascular endothelial cells and may regulate smooth muscle cell (SMC) proliferation through the CDKN1A pathway . Recent research has also identified ANKRD1 as an anti-inflammatory factor with implications in tumor drug resistance mechanisms .

What types of ANKRD1 antibodies are available for research applications?

There are multiple ANKRD1 antibody options available with varying characteristics. Two principal types include polyclonal antibodies like 11427-1-AP (rabbit host) and monoclonal antibodies like 67775-1-Ig (mouse host). These antibodies differ in their specificity, applications, and recommended dilutions as detailed below:

Both antibodies target the same protein with a calculated molecular weight of 319 amino acids (36 kDa) but offer different experimental versatility depending on research requirements .

What are the optimal dilution ratios for different experimental applications?

Appropriate dilutions vary by application and specific antibody used. Based on validated research protocols:

For 11427-1-AP (Polyclonal):

• Western Blot: 1:500-1:3000

• Immunofluorescence (IF-P): 1:200-1:800

For 67775-1-Ig (Monoclonal):

• Western Blot: 1:5000-1:50000 (significantly more concentrated)

• Immunofluorescence (IF/ICC): 1:1000-1:4000

It is strongly recommended to titrate these antibodies in each testing system to obtain optimal results, as performance can be sample-dependent .

How can researchers validate ANKRD1 antibody specificity?

Verification of antibody specificity requires multiple complementary approaches:

• Western blot analysis using positive controls from tissues with known high ANKRD1 expression (e.g., mouse skeletal muscle, mouse heart tissue, rabbit heart tissue)

• Comparison with knockout/knockdown models to confirm band absence

• Peptide competition assay to demonstrate signal specificity

• Cross-validation using alternative antibodies targeting different epitopes of ANKRD1

• Verification of observed molecular weight (36 kDa for ANKRD1)

Published literature shows valid detection in mouse skeletal muscle tissue and rat skeletal muscle tissue for 11427-1-AP, and mouse/rabbit/pig heart tissue for 67775-1-Ig, providing reliable positive controls .

What are the key considerations for successful immunoprecipitation with ANKRD1 antibodies?

Immunoprecipitation with ANKRD1 antibodies requires specific optimization:

• Antibody selection: The polyclonal 11427-1-AP has demonstrated success in IP applications as evidenced by published research

• Lysate preparation: Ensure gentle lysis conditions to preserve protein-protein interactions involving ANKRD1

• Antibody amount: Typically 2-5 μg per 500 μg of total protein lysate

• Pre-clearing: Implement a pre-clearing step with protein A/G beads to reduce non-specific binding

• Negative controls: Include an isotype control antibody (e.g., normal rabbit IgG) processed identically

• Elution conditions: Optimize between gentle (competitive peptide) and denaturing (SDS buffer) methods depending on downstream applications

IP applications are particularly valuable for studying ANKRD1 protein interactions and post-translational modifications.

How can RNA immunoprecipitation (RIP) be performed with ANKRD1 antibodies?

RIP analysis using ANKRD1 antibodies can identify RNA-protein interactions:

• Cell preparation: Cross-link cells with formaldehyde to stabilize RNA-protein complexes

• Cell lysis: Use RNase-free buffers containing RNase inhibitors

• Immunoprecipitation: The 11427-1-AP antibody has been validated for RIP applications

• RNA purification: Extract RNA from immunoprecipitated complexes using standard protocols

• Analysis: Perform RT-PCR or RNA-seq to identify ANKRD1-associated RNAs

This technique has been cited in publications using the 11427-1-AP antibody, indicating its suitability for investigating ANKRD1's role in RNA regulation .

How is ANKRD1 expression altered across different cancer types?

ANKRD1 expression shows significant dysregulation across multiple cancer types, though with notable tissue-specific patterns:

Upregulated in:

• Cholangiocarcinoma (CHOL)

• Glioblastoma multiforme (GBM)

• Liver hepatocellular carcinoma (LIHC)

• Pancreatic adenocarcinoma (PAAD)

• Stomach adenocarcinoma (STAD)

• Uterine corpus endometrial carcinoma (UCEC)

• Uterine carcinosarcoma (UCS)

Downregulated in:

• Colon adenocarcinoma (COAD)

• Head and neck squamous cell carcinoma (HNSC)

• Lung adenocarcinoma (LUAD)

• Lung squamous cell carcinoma (LUSC)

• Rectum adenocarcinoma (READ)

• Skin cutaneous melanoma (SKCM)

• Thyroid carcinoma (THCA)

These differential expression patterns suggest tissue-specific roles for ANKRD1 in carcinogenesis and potential value as a diagnostic biomarker.

What is the prognostic significance of ANKRD1 expression in cancer?

ANKRD1 expression demonstrates significant prognostic value that varies by cancer type:

These findings highlight the context-dependent nature of ANKRD1's role in cancer progression and the importance of tissue-specific research when exploring its potential as a biomarker.

How does ANKRD1 expression correlate with immune cell infiltration in tumors?

ANKRD1 expression demonstrates significant correlations with specific immune cell populations across different cancer types:

• Macrophage infiltration:

  • Positive correlation in liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC)

  • Specifically, M2 macrophage infiltration positively correlates with ANKRD1 expression in LIHC, LUAD, LUSC, SKCM, TGCT, and THCA

• Cancer-associated fibroblasts:

  • Positive correlation in COAD, HNSC, KIRC, LIHC, PRAD, TGCT, and THCA

• Other immune cells:

  • Positive correlation with myeloid dendritic cells in BLCA and PRAD

  • Positive correlation with neutrophils in BRCA, PRAD, and THCA

  • Negative correlation with CD8+ T cells in HNSC and B cells in TGCT

These findings suggest ANKRD1 may play a role in shaping the tumor immune microenvironment, particularly in relation to macrophage recruitment and polarization.

What signaling pathways interact with ANKRD1 in cancer and cardiac tissue?

ANKRD1 is involved in diverse molecular pathways with context-dependent functions:

In cancer:

• Inflammatory and immune pathways in COAD, GBM, and LUSC

• Regulation of SMC proliferation through the CDKN1A pathway

• Association with tumor drug resistance mechanisms

In cardiac tissue:

• Response to physiologic stress, injury, and hypertrophy

• Nuclear transcription repression regulating cardiac gene expression

• Cardiac function pathways in HNSC

These pathway interactions suggest ANKRD1 as a multifunctional protein involved in both tissue-specific homeostasis and pathological processes.

What are common technical challenges when using ANKRD1 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with ANKRD1 antibodies:

• Variable sensitivity across applications:

  • Solution: Optimize antibody concentration for each specific application; the recommended dilution ranges differ significantly between Western blot (1:500-1:3000 for polyclonal; 1:5000-1:50000 for monoclonal) and immunofluorescence (1:200-1:800 for polyclonal; 1:1000-1:4000 for monoclonal)

• Cross-reactivity with related proteins:

  • Solution: Include appropriate negative controls; validate with tissues known to be negative for ANKRD1 expression

• Background signal in immunofluorescence:

  • Solution: Implement additional blocking steps; optimize antibody concentration; include proper washing steps between incubations

• Sample-dependent variations:

  • Solution: Test antibodies on multiple samples and validate with positive controls (e.g., mouse skeletal muscle tissue, mouse heart tissue)

How should ANKRD1 antibodies be stored to maintain optimal performance?

Proper storage is critical for maintaining antibody performance:

• Storage temperature: Store at -20°C

• Storage buffer: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability: Stable for one year after shipment when stored properly

• Aliquoting: For most applications, aliquoting is unnecessary for -20°C storage

• Special considerations: Small volume formats (20μl) may contain 0.1% BSA as a stabilizer

Adhering to these storage conditions will help ensure reproducible results across experiments.

How can ANKRD1 antibodies contribute to therapeutic development in cancer?

ANKRD1 antibodies can facilitate several approaches to therapeutic development:

• Biomarker validation: ANKRD1 shows potential as a prognostic biomarker, particularly in COAD where it serves as an independent prognostic factor

• Drug sensitivity assessment: ANKRD1 may influence the half-maximal inhibitory concentration (IC50) of several anti-tumor drugs, suggesting a role in predicting treatment response

• Target validation: In vitro experiments demonstrate that ANKRD1 promotes migration and invasion while inhibiting apoptosis in colorectal cancer cell lines (Caco2, SW480)

• Immune therapy connections: ANKRD1's correlations with immune cell infiltration and immune checkpoints suggest potential applications in immunotherapy research

Understanding these aspects could lead to novel therapeutic strategies targeting ANKRD1 or its associated pathways.

What novel experimental approaches are being developed to study ANKRD1 function?

Emerging research methodologies for ANKRD1 investigation include:

• Combined multi-omics approaches integrating:

  • Transcriptomics (mRNA expression)

  • Epigenomics (DNA methylation)

  • Proteomics (protein-protein interactions)

  • Genomics (mutation status)

• Advanced functional studies:

  • Gene Set Variation Analysis (GSVA) to identify enriched pathways

  • Integration with immune infiltration data

  • Correlation with tumor mutational burden (TMB), microsatellite instability (MSI), and mismatch repair (MMR) status

• Clinical correlation studies linking ANKRD1 expression with:

  • Treatment response

  • Patient outcomes across multiple cancer types

  • Immune checkpoint expression

These approaches will likely yield more comprehensive insights into ANKRD1's diverse biological functions and therapeutic potential.