CDKN1B (Ab-10) Antibody

What is the molecular target of CDKN1B (Ab-10) Antibody and what epitope does it recognize?

The CDKN1B (Ab-10) Antibody specifically targets the cyclin-dependent kinase inhibitor 1B protein (p27Kip1), recognizing a peptide sequence around amino acids 8-12 (N-G-S-P-S) derived from Human p27Kip1 . This antibody detects endogenous levels of total p27Kip1 protein with high specificity . The p27Kip1 protein functions as a key negative regulator of cell cycle progression from G1 to S phase by controlling the function of multiple cyclin-dependent kinases (CDKs)/cyclin complexes . For optimal detection, researchers should be aware that this N-terminal epitope may be affected by post-translational modifications that could alter antibody recognition.

What applications is CDKN1B (Ab-10) Antibody validated for?

The CDKN1B (Ab-10) Antibody has been validated for multiple applications including ELISA, Western Blotting (WB), and Immunohistochemistry (IHC) . For Western Blotting, the recommended dilution range is 1:500-1:1000, while for Immunohistochemistry, a dilution range of 1:50-1:200 is suggested . When designing experiments, it's important to include appropriate positive controls. For example, the antibody has been validated using extracts from A2780 cells for Western blotting and human lung carcinoma tissue for immunohistochemistry .

What species reactivity does CDKN1B (Ab-10) Antibody demonstrate?

The CDKN1B (Ab-10) Antibody exhibits reactivity against Human, Mouse, and Rat samples . When working with animal models, it's important to note that while CDKN1B is highly conserved across these species, subtle sequence variations may exist. For comparative studies across different model organisms, it's advisable to perform a sequence alignment analysis to confirm epitope conservation and validate antibody performance with species-specific positive controls.

How can researchers optimize CDKN1B (Ab-10) Antibody for immunohistochemistry applications?

For immunohistochemistry applications, researchers should consider the following optimization protocol:

• Antigen retrieval: When working with formalin-fixed tissues, heating tissue sections in 10 mM Tris with 1 mM EDTA, pH 9.0, for 45 min at 95°C followed by cooling at room temperature for 20 minutes may be necessary for optimal epitope exposure .

• Antibody concentration: Begin with the recommended dilution of 1:50-1:200 and optimize based on your specific tissue type .

• Detection system: For chromogenic detection, a biotinylated secondary antibody (1:1000) followed by peroxidase streptavidin (1:500) has been successfully used .

• Controls: Include positive controls such as tonsil, breast, cervical or colon carcinoma tissues which are known to express p27Kip1 .

• Signal interpretation: CDKN1B staining patterns should be evaluated in context of cell cycle markers, with particular attention to nuclear versus cytoplasmic localization which may indicate different functional states of the protein.

What are the considerations for studying CDKN1B loss-of-function variants in disease models?

When investigating CDKN1B loss-of-function variants, researchers should employ a comprehensive approach:

• Genetic screening: Utilize germline whole-exome sequencing (WES) and/or Sanger sequencing to identify potential pathogenic variants .

• Variant validation: For putative pathogenic variants, conduct functional characterization to determine their effects on protein stability and function .

• Expression analysis: Employ CDKN1B (Ab-10) Antibody to assess protein levels and localization in affected tissues.

• Functional domains: Pay particular attention to variants affecting the CDKN1B protein scatter domain (amino acids 119-136), which has been implicated in pathogenic variants (p.I119T, p.E126Q, and p.D136G) .

• Animal models: Consider that Cdkn1b knockout mice develop ACTH-secreting hyperplasia or adenomas of the pituitary pars intermedia with full penetrance, providing a valuable model system .

How can researchers verify CDKN1B (Ab-10) Antibody specificity?

To ensure experimental validity, researchers should verify antibody specificity through multiple approaches:

• Knockout/knockdown controls: Test the antibody in CDKN1B knockout or knockdown samples to confirm signal specificity.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate specific blocking of the signal.

• Multiple antibodies: Compare staining patterns with other validated CDKN1B antibodies targeting different epitopes to confirm consistency.

• Expected molecular weight: Verify that the detected protein corresponds to the expected molecular weight of 27 kDa for p27Kip1 .

• Cross-reactivity assessment: Confirm that the antibody shows no cross-reaction with other related mitotic inhibitors .

What are common challenges when using CDKN1B (Ab-10) Antibody for Western blotting?

When performing Western blotting with CDKN1B (Ab-10) Antibody, researchers may encounter several challenges:

• Multiple bands: Besides the expected 27 kDa band, additional bands may represent post-translationally modified forms of CDKN1B or non-specific binding. Validation with knockout controls and phosphatase treatment can help distinguish these possibilities.

• Weak signal: If signal is weak despite appropriate loading controls, consider:

  • Increasing antibody concentration (up to 1:500)

  • Extending incubation time (overnight at 4°C)

  • Using enhanced chemiluminescence detection systems

  • Verifying sample handling to prevent protein degradation

• High background: To reduce background staining:

  • Optimize blocking conditions (5% BSA may be preferable to milk for phospho-epitopes)

  • Increase washing duration and frequency

  • Filter antibody solutions before use

  • Reduce secondary antibody concentration

• Inconsistent results: For reproducible results, standardize lysate preparation methods and use positive control lysates from recommended cell lines such as ZR75, T47D, SK-BR-3, MDA-MB-231, HeLa or MCF7 cells .

How should researchers interpret CDKN1B expression in correlation with cell cycle status?

Interpretation of CDKN1B expression requires understanding its role in cell cycle regulation:

• Normal expression patterns: CDKN1B is highly expressed in all hormone-producing cells of the anterior pituitary, with corticotrophs displaying the lowest levels .

• Expression in neoplasms: CDKN1B levels are often significantly reduced in pituitary neuroendocrine tumors (PitNETs), and very weak nuclear CDKN1B staining is frequently found in corticotropinomas .

• Subcellular localization: Nuclear versus cytoplasmic localization is critical - nuclear CDKN1B primarily functions as a CDK inhibitor, while cytoplasmic localization may indicate alternative functions or inactivation.

• Correlation with proliferation markers: CDKN1B staining inversely correlates with Ki-67 expression, providing a valuable measure of proliferative activity .

• Cell synchronization studies: For meaningful interpretation, consider cell cycle phase when analyzing CDKN1B levels, ideally with synchronized cell populations or co-staining for cell cycle phase markers.

What experimental controls are essential when using CDKN1B (Ab-10) Antibody?

Rigorous experimental design requires appropriate controls:

• Positive controls: Use cell lines known to express CDKN1B, such as A2780 cells for Western blot or human lung carcinoma tissue for IHC .

• Negative controls: Include samples where the primary antibody is omitted but all other steps are identical.

• Isotype controls: Use irrelevant antibodies of the same isotype and concentration to identify non-specific binding.

• Loading controls: For Western blotting, include appropriate housekeeping proteins to normalize for loading variations.

• Biological validation: Correlate CDKN1B expression with expected biological outcomes, such as cell cycle arrest or response to TGF-β treatment.

How can CDKN1B (Ab-10) Antibody be used in co-immunoprecipitation studies?

While not explicitly mentioned in the product applications, researchers may consider using this antibody for co-immunoprecipitation to study CDKN1B interactions:

• Lysate preparation: Use gentle lysis buffers (e.g., 10 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40) to preserve protein-protein interactions.

• Antibody coupling: Consider pre-coupling the antibody to protein A/G beads to avoid co-elution of IgG chains that may interfere with Western blot detection.

• Known interactions: Design experiments to validate known interactions, such as CDKN1B complexes with CDK4, as this antibody has been shown to co-precipitate CDK4 in complex with p27Kip1 .

• Elution conditions: Optimize elution conditions to efficiently release complexes while maintaining their integrity.

• Validation approaches: Confirm interactions through reciprocal co-IPs and alternative methods such as proximity ligation assays.

What approaches can researchers use to study post-translational modifications of CDKN1B?

Investigating post-translational modifications of CDKN1B requires specialized techniques:

• Phosphorylation analysis: Since CDKN1B function is regulated by phosphorylation at multiple sites (including Thr187), researchers should:

  • Use phospho-specific antibodies in parallel with CDKN1B (Ab-10) Antibody

  • Treat samples with phosphatase inhibitors during preparation

  • Consider phosphatase treatment controls to confirm specificity

  • Employ Phos-tag gels to separate phosphorylated from non-phosphorylated forms

• Ubiquitination studies: To study degradation pathways:

  • Treat cells with proteasome inhibitors (e.g., MG132)

  • Use denaturing lysis conditions to preserve ubiquitin linkages

  • Immunoprecipitate CDKN1B and probe for ubiquitin or vice versa

• Subcellular fractionation: To analyze localization-dependent modifications:

  • Separate nuclear and cytoplasmic fractions before immunoblotting

  • Correlate modifications with subcellular localization and function

How can CDKN1B (Ab-10) Antibody contribute to research on endocrine neoplasia?

Given the role of CDKN1B mutations in multiple endocrine neoplasia type 4 (MEN4), this antibody offers valuable research applications:

• Clinical correlation: Compare CDKN1B expression levels in tissue samples from patients with and without germline CDKN1B variants .

• Functional characterization: Use the antibody to assess protein stability and localization of mutant CDKN1B proteins in cell models .

• Domain-specific effects: Pay particular attention to variants affecting the scatter domain (amino acids 119-136) as these have been implicated in pathogenic variants (p.I119T, p.E126Q, and p.D136G) .

• Tissue-specific expression: Compare CDKN1B expression across different endocrine tissues to understand tissue-specific susceptibility to tumorigenesis.

• Therapeutic implications: Monitor CDKN1B expression changes in response to potential therapeutic interventions targeting the cell cycle in endocrine neoplasias.