CDKN2AIP Antibody

What is CDKN2AIP and why is it important in research?

CDKN2AIP is an RNA-binding protein that plays critical roles in stem cell pluripotency, somatic differentiation, and spermatogenesis. Research has demonstrated that CDKN2AIP regulates DNA damage response in a dose-dependent manner through multiple signaling pathways involved in cell proliferation, apoptosis, and senescence .

CDKN2AIP was initially discovered as a novel ARF-binding protein that can interact with p53 both directly and ARF-independently. Its highest expression is found in testicular tissue, where it appears to function as a tumor suppressor . Recent studies have revealed its importance in:

• Spermiogenesis and germ cell development

• Activation of the Wnt-signaling pathway

• DNA damage response regulation

• Tumor suppression, particularly in testicular seminomas

What experimental applications are CDKN2AIP antibodies suitable for?

Based on commercially available antibodies and research publications, CDKN2AIP antibodies have been validated for multiple experimental applications:

What is the molecular weight of CDKN2AIP and how can I confirm specificity?

CDKN2AIP has a calculated molecular weight of approximately 61 kDa, though it's often observed at around 65 kDa in Western blot applications . To confirm antibody specificity:

• Use positive controls like 293T, HeLa, or Jurkat whole cell lysates, which show consistent CDKN2AIP expression

• Include negative controls such as CDKN2AIP knockout cell lines when available

• Verify band size corresponds to the predicted molecular weight

• Test cross-reactivity with related proteins in the CDKN2 family

How should I design experiments to study CDKN2AIP interactions with other proteins?

When investigating protein-protein interactions involving CDKN2AIP, consider these methodological approaches:

• Co-immunoprecipitation followed by mass spectrometry (IP-MS):

  • Use CDKN2AIP antibody incubated with cell/tissue lysate (4°C for 6 hours)

  • Add protein A beads and incubate overnight with gentle shaking at 4°C

  • Wash bead complexes with incubation buffer (3 × 5 minutes at 4°C)

  • Elute protein complexes with SDS loading buffer

  • Visualize by silver staining and analyze by mass spectrometry

• Biolayer interferometry (BLI):

  • Express CDKN2AIP as a His-fusion protein

  • Dilute in appropriate assay buffer (e.g., 50 mM Tris buffer pH 7.2, 25 mM NaCl)

  • Perform binding assays at controlled temperature (30°C) with agitation (1000 rpm)

• Verification of interactions:
  Research has identified several CDKN2AIP-interacting proteins including CARM1, eIF4β, SEC24C, CTTN, HBB, NCL, and POLR2A, which can serve as positive controls .

What controls should I include when studying CDKN2AIP expression in normal versus cancer tissues?

Based on published methodologies, include the following controls:

• Positive tissue controls:

  • Normal testis tissue (shows highest CDKN2AIP expression)

  • Cell lines with confirmed CDKN2AIP expression (293T, HeLa, Jurkat, NIH3T3)

• Negative controls:

  • CDKN2AIP knockout or knockdown cells

  • Tissues with minimal CDKN2AIP expression

  • Isotype control antibodies (e.g., control rat IgG for IP experiments)

• Comparative analysis:
  Compare expression across multiple tissue types to establish relative expression levels, as research has shown differential expression patterns .

• Technical validation:

  • Use multiple detection methods (WB, IHC, qRT-PCR)

  • Include housekeeping genes/proteins (β-actin, GAPDH) as loading controls

How can I optimize Western blot detection of CDKN2AIP?

For optimal Western blot results with CDKN2AIP antibodies:

• Sample preparation:

  • Use RIPA or IP buffer with protease inhibitors for cell/tissue lysis

  • Centrifuge at 12,000 rpm for 30 minutes at 4°C to remove debris

• Antibody conditions:

  • Use dilutions between 0.04-0.4 μg/mL for commercial antibodies

  • Add 0.1% Tween 20 to blocking buffer and primary antibody incubation buffer

  • Incubate primary antibody overnight at 4°C

• Detection optimization:

  • Use ECL technique with 30-second exposure as a starting point

  • Load appropriate protein amounts (15-50 μg) depending on expression level

  • Consider enhanced chemiluminescence methods for low expression samples

• Common issues and solutions:

  • Multiple bands: Use CDKN2AIP knockout controls to identify specific bands

  • Weak signal: Increase antibody concentration or protein loading

  • High background: Increase washing steps or blocking time

What are the best methods for studying CDKN2AIP function in gene regulation?

Based on research methodologies, consider these approaches:

• Gene expression analysis:

  • Extract total RNA using TRIzol reagent

  • Perform RT-PCR with β-actin as a housekeeping gene

  • Design specific primers for CDKN2AIP and related genes (e.g., SUN1)

• Functional knockdown studies:

  • Use CDKN2AIP CRISPR/Cas9 KO plasmids for gene knockout

  • Design gRNAs targeting 5' constitutive exons of CDKN2AIP

  • Verify knockout by Western blot and RT-PCR

• Cell cycle and apoptosis analysis:

  • Measure cell senescence using SA-β-gal staining assay

  • Evaluate H3K9me3 activity for senescence-associated heterochromatin foci

  • Analyze DNA damage using DSB (double-strand break) markers

How does CDKN2AIP expression change during tumor progression, and what are the implications for cancer research?

Research findings demonstrate complex patterns of CDKN2AIP expression in cancer:

• Expression patterns:

  • Significantly lower in testicular seminoma tissues compared to normal tissues

  • Expression changes correlate with tumor cell senescence and apoptosis

  • CDKN2AIP amplification observed in invasive and metastatic malignancies

• Functional consequences:

  • Moderate CDKN2AIP upregulation causes growth arrest and senescence

  • Excessive enrichment facilitates aggressive proliferation and malignant transformation

  • CDKN2AIP promotes epithelial-mesenchymal transition (EMT) through Wnt/β-catenin activation

• Signaling pathways:

  • CDKN2AIP interacts with CARM1 and eIF4β in testicular seminoma

  • CDKN2AIP-induced tumor cell senescence occurs through suppression of CARM1 expression and eIF4β phosphorylation

  • These CDKN2AIP interactions represent potential druggable pathways

How can CDKN2AIP antibodies be used to study the protein's role in spermatogenesis and male fertility?

CDKN2AIP plays critical roles in male germ cell development:

• Experimental approaches:

  • Immunofluorescence microscopy to localize CDKN2AIP in different cell types (spermatogonia, spermatocytes, spermatids)

  • Compare CDKN2AIP+/+ and CDKN2AIP-/- mice to analyze sperm morphology and fertility

  • Evaluate synapsis, DNA repair, and protamine replacement using CDKN2AIP antibodies

• Key research findings:

  • CDKN2AIP is expressed in spermatocytes and spermatids

  • CDKN2AIP-/- mice exhibit multiple sperm head defects and age-dependent germ cell loss

  • Loss of CDKN2AIP causes synapsis failure in ~19% of spermatocytes and increased apoptosis

  • CDKN2AIP knockdown is associated with extended S phase, increased DNA damage, and apoptosis

What are the methodological considerations for studying CDKN2AIP's role in DNA damage response pathways?

When investigating CDKN2AIP in DNA damage response:

• Experimental design:

  • Create dose-dependent expression models (knockdown, normal, and overexpression)

  • Induce DNA damage using standardized methods (radiation, chemical agents)

  • Evaluate cellular responses through multiple endpoints (cell cycle analysis, apoptosis markers, DNA repair proteins)

• Mechanisms to investigate:

  • ATR/CHK1 pathway activation following CDKN2AIP knockdown

  • p53-HDM2-p21 pathway in CDKN2AIP overexpression conditions

  • CDKN2AIP interactions with DNA repair machinery

• Research implications:

  • Knocking down CDKN2AIP results in aneuploidy, DNA damage, and mitotic catastrophe

  • CDKN2AIP overexpression impairs cell proliferation and induces senescence

  • Understanding these pathways may reveal potential therapeutic targets for cancer treatment

How can mixed-method research designs enhance our understanding of CDKN2AIP function?

Integration of qualitative and quantitative methodologies offers comprehensive insights:

• Quantitative approaches:

  • Measure CDKN2AIP expression levels across tissues and disease states

  • Evaluate statistical associations between expression and clinical outcomes

  • Test specific hypotheses regarding CDKN2AIP-related pathways

• Qualitative approaches:

  • Characterize the nature of protein-protein interactions

  • Describe subcellular localization patterns in different cell types

  • Explore mechanistic aspects of CDKN2AIP function

• Mixed-method implementation:

  • Use quantitative methods to establish expression patterns and correlations

  • Apply qualitative approaches to determine mechanism and function

  • Integrate findings to develop comprehensive models of CDKN2AIP activity

What are the latest methodological advances for studying CDKN2AIP in cancer metastasis and EMT?

Recent research has revealed CDKN2AIP's role in cancer progression:

• Advanced techniques:

  • In vivo targeting using naked siRNA or CDKN2AIP shRNA in adeno-oncolytic viruses

  • Monitoring of tumor progression and metastasis in animal models

  • Analysis of nuclear β-catenin localization and downstream effectors

• Key findings:

  • CDKN2AIP enrichment activates the Wnt/β-catenin signaling axis

  • Enhanced nuclear localization of β-catenin and increased levels of SNAIL1, SNAIL2, ZEB1, and TWIST1

  • Targeted CDKN2AIP knockdown reduces nuclear β-catenin and EMT effectors

• Research implications:

  • CDKN2AIP represents a potential therapeutic target for aggressive cancers

  • Inhibiting CDKN2AIP-mediated EMT could suppress tumor invasiveness and metastasis

  • Understanding these mechanisms may lead to novel treatment strategies