CDK2AP1 Antibody

What is the optimal concentration range for using CDK2AP1 antibodies in different applications?

Based on multiple commercial antibody specifications, the following dilution ranges are recommended for CDK2AP1 antibodies:

For optimal results, it is recommended to titrate the antibody in each specific testing system. Sensitivity can vary depending on cell types and tissue preparation methods. The Sigma-Aldrich HPA057648 antibody specifically recommends 0.25-2 μg/mL for immunofluorescence applications .

What are the different available forms of CDK2AP1 antibodies and their specific research applications?

Multiple forms of CDK2AP1 antibodies are available, each with distinct characteristics suitable for different experimental objectives:

The choice between these antibodies depends on your experimental needs. Polyclonal antibodies generally offer higher sensitivity but potentially lower specificity compared to monoclonal alternatives. For co-immunoprecipitation studies, affinity-isolated antibodies may provide better results.

What antigen targets are used to generate CDK2AP1 antibodies?

CDK2AP1 antibodies are generated using various immunogen sequences targeting different regions of the protein:

• Full-length protein: Some antibodies like Proteintech's 13060-2-AP use the complete CDK2AP1 protein sequence (1-115 amino acids encoded by BC034717) .

• Partial sequences: Sigma-Aldrich's HPA057648 uses the sequence "ATSSQYRQLLSDYGPPSLGYTQGTGNSQVPQSKYAELLAIIEELGKEI" .

• Fusion proteins: Several manufacturers use fusion protein constructs to generate antibodies with specific epitope recognition.

When selecting an antibody, consider which protein region is most relevant to your research questions. For instance, if studying CDK2AP1 dimerization, antibodies recognizing the region around Cys-105 might be most informative, as this residue is critical for disulfide bond formation and biological activity .

How can I validate the specificity of a CDK2AP1 antibody for my particular application?

Comprehensive validation of CDK2AP1 antibodies should include multiple approaches:

• Positive and negative control samples: Use cell lines with known CDK2AP1 expression levels. HEK-293 and HeLa cells have been verified as positive controls for Western blot analysis .

• Knockdown/knockout validation: Compare antibody reactivity in wildtype versus CDK2AP1-knockdown/knockout cells. This is particularly important as CDK2AP1 has multiple aliases and isoforms.

• Expected molecular weight verification: CDK2AP1 has a calculated molecular weight of approximately 13 kDa but is typically observed at 12 kDa in SDS-PAGE .

• Cross-reactivity testing: If working with non-human samples, verify species cross-reactivity, as not all CDK2AP1 antibodies react with mouse or rat proteins despite high sequence conservation.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal specificity.

For detecting both monomeric and dimeric forms of CDK2AP1, ensure your sample preparation preserves or disrupts disulfide bonds as appropriate to your research questions. The dimeric form (linked by Cys-105 disulfide bonds) has been identified as the active form in contact-inhibited human diploid cells .

What are the best sample preparation methods when working with CDK2AP1 antibodies?

Optimal sample preparation depends on your specific application:

• For Western blotting:

  • Use RIPA buffer with protease inhibitors for cell lysate preparation

  • Include phosphatase inhibitors if studying CDK2AP1 phosphorylation status

  • For detecting both monomeric and dimeric forms, run samples under both reducing and non-reducing conditions

  • Avoid excessive sample heating which can cause protein aggregation

• For immunohistochemistry:

  • Formalin-fixed paraffin-embedded (FFPE) tissues work well with most CDK2AP1 antibodies

  • Antigen retrieval: Multiple protocols recommend TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative

  • Background reduction: Block with appropriate serum (typically 5-10% normal serum from the secondary antibody host species)

• For immunofluorescence:

  • 4% paraformaldehyde fixation is typically effective

  • Permeabilize with 0.1-0.5% Triton X-100

  • Recommended dilution ranges from 1:50-1:500, optimized for your specific application

For all applications, include appropriate controls and standardize protocols to ensure reproducibility.

How can I distinguish between different CDK2AP1 isoforms with antibodies?

CDK2AP1 has multiple transcript variants encoding distinct isoforms due to alternative splicing . To distinguish between these:

• Select antibodies with mapped epitopes: Choose antibodies targeting regions that differ between isoforms.

• Use isoform-specific positive controls: Express individual isoforms in cell lines lacking endogenous CDK2AP1.

• Combine with RT-PCR: Complement antibody-based detection with transcript-specific PCR to confirm isoform expression patterns.

• 2D gel electrophoresis: Combine with Western blotting to separate isoforms based on both molecular weight and isoelectric point.

• Mass spectrometry validation: For definitive identification, combine immunoprecipitation with mass spectrometry.

How can I use CDK2AP1 antibodies to study its role in cancer progression and as a potential biomarker?

CDK2AP1 has emerged as an important cancer-related protein with context-dependent roles. Research applications include:

• Expression analysis in clinical samples:

  • Use IHC to analyze CDK2AP1 expression in tumor microarrays

  • Correlate expression with clinical outcomes, as high CDK2AP1 expression has been associated with poor prognosis in hepatocellular carcinoma

  • Compare expression between tumor and adjacent normal tissues

• Studying molecular mechanisms:

  • Use co-immunoprecipitation with CDK2AP1 antibodies to identify interaction partners

  • Examine cell cycle regulation by analyzing CDK2 activity in relation to CDK2AP1 expression

  • Investigate the role of CDK2AP1 in DNA replication through its interaction with DNA polymerase alpha/primase

• Immune infiltration studies:

  • CDK2AP1 expression shows positive correlations with infiltrating immune cells in HCC, including B cells, CD4+ T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells

  • Combine CDK2AP1 IHC with immune cell markers for multiplex analysis

• microRNA regulation:

  • Several microRNAs (miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p) have been identified to inhibit CDK2AP1 translation in oral squamous cell carcinomas

  • Use antibodies to measure protein levels while simultaneously assessing miRNA expression

What are the appropriate experimental designs to study CDK2AP1's dual role in cell cycle regulation and epigenetic control?

CDK2AP1 functions in both cell cycle regulation and as part of the NuRD complex for epigenetic regulation. To investigate these distinct roles:

• Cell cycle regulation studies:

  • Synchronize cells and analyze CDK2AP1 expression/localization across cell cycle phases

  • Use proximity ligation assays with CDK2AP1 and CDK2 antibodies to visualize interactions in situ

  • Combine with CDK2 activity assays to correlate CDK2AP1 binding with kinase inhibition

  • Use non-reducing conditions to preserve the active disulfide-bonded dimeric form

• Epigenetic regulation studies:

  • Perform chromatin immunoprecipitation (ChIP) using CDK2AP1 antibodies to identify genomic binding sites

  • Co-immunoprecipitate with other NuRD complex components

  • Analyze histone modifications at CDK2AP1-bound regions

  • Study CDK2AP1's role in embryonic stem cell differentiation through conditional knockout systems

• Integrated approaches:

  • Use proximity-dependent biotinylation (BioID) with CDK2AP1 as bait to identify context-specific interaction partners

  • Perform nucleocytoplasmic fractionation to determine compartment-specific functions

  • Develop separation-of-function mutants that disrupt either cell cycle or epigenetic regulatory roles

When designing these experiments, consider the timing and cellular context, as CDK2AP1's functions may vary depending on cell type, differentiation state, and cell cycle phase.

How can I integrate CDK2AP1 antibody-based techniques with genomic and proteomic approaches for comprehensive pathway analysis?

For comprehensive pathway analysis, combine antibody-based techniques with multi-omics approaches:

• Antibody-based proteomics integration:

  • Use CDK2AP1 immunoprecipitation followed by mass spectrometry to identify interactome

  • Perform reverse phase protein arrays (RPPA) to analyze signaling pathways affected by CDK2AP1 modulation

  • Apply proximity-dependent biotinylation (BioID) or APEX tagging with CDK2AP1 as bait

• Genomic integration:

  • Combine ChIP-seq using CDK2AP1 antibodies with RNA-seq to correlate binding with gene expression changes

  • Integrate with ATAC-seq to assess chromatin accessibility at CDK2AP1-regulated regions

  • Analyze CDK2AP1 binding at specific cell cycle gene promoters

• Functional assays:

  • Study pathways identified in KEGG analysis, including Fc gamma R-mediated phagocytosis, Th1 and Th2 cell differentiation, and cell cycle regulation

  • Validate pathway connections using pharmacological inhibitors or CRISPR-based approaches

This integrated approach has revealed CDK2AP1's involvement in immune environment regulation in hepatocellular carcinoma and its potential as an immunotherapy target .

How do I reconcile the conflicting roles of CDK2AP1 as both tumor suppressor and oncogene in different cancer contexts?

The literature presents an apparent paradox regarding CDK2AP1's role in cancer:

• Tumor suppressor evidence:

  • Originally identified as deleted in oral cancer (DOC1)

  • Reduced expression in malignant oral keratinocytes

  • Inhibits cell growth when transformed keratinocytes are transfected with doc-1

  • Negative regulator of CDK2 activity

• Oncogenic evidence:

  • Higher expression in hepatocellular carcinoma compared to normal liver tissues

  • Positive correlation with advanced clinical stage and histologic grade in HCC patients

  • Poor prognosis associated with high CDK2AP1 expression in HCC

  • Promotion of proliferation and metastasis in HCC

To reconcile these conflicting roles, consider:

• Tissue context: CDK2AP1 function may be tissue-specific, acting as a suppressor in oral tissues but an oncogene in liver tissues

• Molecular context: Different interaction partners in various tissues may determine function

• Genetic background: The effect of CDK2AP1 may depend on the status of other tumor suppressors/oncogenes

• Epigenetic versus cell cycle roles: The dominant function may vary by context

• Isoform expression: Different splice variants may have opposing functions

When designing experiments, include multiple tissue types and measure both cell cycle and epigenetic regulatory outcomes to determine the predominant role in your specific system.

What are the latest methodological advances in studying CDK2AP1 protein-protein interactions and functional dynamics?

Recent technological advances have enhanced our ability to study CDK2AP1:

• Structural insights:

  • Crystal structure studies have revealed CDK2AP1 forms a four-helix dimeric structure

  • The disordered N-terminal region may facilitate dynamic interactions with multiple partners

• Advanced microscopy techniques:

  • Super-resolution microscopy enables visualization of CDK2AP1 co-localization with interaction partners at nanoscale resolution

  • Live-cell imaging with fluorescently tagged CDK2AP1 allows real-time tracking of its dynamics during cell cycle progression

• Proximity-based interaction mapping:

  • BioID and TurboID approaches with CDK2AP1 as bait can identify transient and stable interaction partners

  • APEX2-based proximity labeling provides temporal resolution of interaction networks

• Single-molecule techniques:

  • Fluorescence correlation spectroscopy (FCS) to measure CDK2AP1 diffusion and binding kinetics

  • Single-molecule pull-down (SiMPull) assays to analyze stoichiometry of CDK2AP1-containing complexes

• Novel tissue analysis methods:

  • Combined in situ hybridization (ISH) with immunofluorescence (IF) on tissue microarrays to study miRNA regulation of CDK2AP1 in tumor architectural context

These approaches are revealing new insights into CDK2AP1's dual roles in cell cycle regulation and epigenetic control, particularly its integration with the NuRD complex.

How can I design experiments to investigate the relationship between CDK2AP1 and immune infiltration in the tumor microenvironment?

Based on recent findings linking CDK2AP1 to immune infiltration , consider these experimental approaches:

• Correlation analysis in patient samples:

  • Perform multiplex IHC using CDK2AP1 antibodies alongside immune cell markers

  • Analyze spatial relationships between CDK2AP1-expressing cells and immune infiltrates

  • Stratify patients based on CDK2AP1 expression and compare immune profiles

• Mechanistic studies in cell culture and animal models:

  • Create CDK2AP1 knockout and overexpression models in cancer cell lines

  • Perform co-culture experiments with immune cells to assess direct effects

  • Use conditioned media experiments to identify secreted factors regulated by CDK2AP1

  • Develop syngeneic mouse models with CDK2AP1 modulation to assess in vivo immune recruitment

• Pathway analysis:

  • Investigate the Fc gamma R-mediated phagocytosis and Th1/Th2 cell differentiation pathways identified in KEGG analysis

  • Perform cytokine/chemokine profiling of CDK2AP1-modified cells

  • Use phospho-specific antibodies to map signaling cascades affecting immune regulation

• Clinical correlation:

  • Analyze CDK2AP1 expression in relation to response to immunotherapy

  • Correlate with immune checkpoint expression (PD-1, PD-L1)

  • Develop predictive models incorporating CDK2AP1 status for immunotherapy response

Given that CDK2AP1 expression shows positive correlations with various immune cell infiltrates in HCC (B cells, CD4+ T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells) , it could serve as a potential biomarker for immunotherapy selection.

What are the optimal conditions for immunoprecipitation of CDK2AP1 and its binding partners?

For successful co-immunoprecipitation of CDK2AP1 complexes:

• Lysis buffer optimization:

  • Standard IP buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease and phosphatase inhibitors

  • For preserving weak interactions: Reduce salt concentration to 100 mM NaCl and use milder detergents (0.3% CHAPS)

  • For capturing chromatin-associated complexes: Include nuclease treatment (e.g., Benzonase)

• Antibody selection:

  • Use affinity-purified antibodies like Sigma's HPA057648 or Proteintech's 13060-2-AP

  • Pre-clear lysates with species-matched IgG to reduce non-specific binding

  • Consider epitope-tagged CDK2AP1 constructs for highly specific purification

• Bead considerations:

  • Protein A/G magnetic beads generally provide better results than agarose beads

  • Pre-couple antibody to beads before adding lysate for cleaner results

  • Use low binding tubes to minimize protein loss

• Special considerations for CDK2AP1:

  • To capture dimeric form: Use non-reducing conditions throughout

  • To enrich for CDK2 interaction: Synchronize cells in G1/S transition

  • To study NuRD complex: Include nuclear extraction steps

• Elution strategies:

  • Gentle elution: Use excess immunizing peptide

  • Standard elution: Low pH glycine buffer (0.1 M, pH 2.5) with immediate neutralization

  • For subsequent mass spectrometry: On-bead digestion is recommended

Cross-linking the antibody to beads can reduce antibody contamination in downstream applications like mass spectrometry.

How should I optimize immunohistochemistry protocols for CDK2AP1 detection in different tissue types?

Optimizing IHC for CDK2AP1 across different tissue types requires attention to several variables:

• Fixation considerations:

  • FFPE tissues: 10% neutral buffered formalin fixation for 24-48 hours is standard

  • Frozen sections: Fix with 4% paraformaldehyde for 10-15 minutes prior to staining

• Antigen retrieval optimization:

  • Primary method: TE buffer pH 9.0 with heat treatment

  • Alternative method: Citrate buffer pH 6.0

  • Optimization approach: Test both methods side by side with positive control tissues

• Antibody dilution by tissue type:

  • Start with manufacturer's recommended range (1:50-1:500)

  • For high-expressing tissues (liver, testis): Use higher dilutions

  • For low-expressing tissues: Use lower dilutions

  • Always include positive control tissues with known expression

• Detection system:

  • For lower expression: Use amplification systems (TSA, polymer-based)

  • For multiplex IHC: Consider tyramide signal amplification systems

  • Chromogen selection: DAB provides good contrast but consider others for multiplex work

• Tissue-specific considerations:

  • High background in liver: Additional blocking with avidin/biotin

  • Brain tissue: Extended antigen retrieval may be necessary

  • Skin: Requires careful blocking to prevent edge effect

Validation should include known positive tissues (testis, brain, heart, liver, ovary, placenta, skin, and spleen have been documented to show positive staining) .

What are the considerations for selecting CDK2AP1 antibodies for use in flow cytometry applications?

Although not explicitly mentioned in the search results as a common application, flow cytometry with CDK2AP1 antibodies is possible with proper optimization:

• Antibody selection criteria:

  • Choose antibodies validated for immunofluorescence applications

  • Polyclonal antibodies generally provide stronger signal but potentially higher background

  • Confirm the antibody recognizes native conformations (not just denatured protein)

• Sample preparation:

  • For intracellular staining: Fix with 2-4% paraformaldehyde

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100

  • Optimize fixation and permeabilization time for your specific cell type

• Staining protocol optimization:

  • Starting dilution: Use 2-3× more concentrated than for microscopy

  • Include proper blocking: 1-5% BSA or 5-10% serum matching secondary antibody species

  • Extended incubation (1-2 hours) may improve signal quality

  • Include FcR blocking reagent to reduce non-specific binding

• Controls:

  • Isotype controls matched to primary antibody

  • Positive control: Cell lines with known high CDK2AP1 expression (HeLa, HEK-293)

  • Negative control: CDK2AP1 knockdown/knockout cells

  • Fluorescence minus one (FMO) controls for multicolor panels

• Analysis considerations:

  • CDK2AP1 is primarily intracellular, requiring permeabilization

  • May show cell cycle-dependent expression patterns

  • Consider analyzing correlation with cell cycle markers (e.g., Ki-67, cyclins)