CDK5RAP3 Antibody

What is CDK5RAP3 and why is it important in research?

CDK5RAP3 (CDK5 regulatory subunit associated protein 3) was initially identified as a binding protein of the CDK5 activator p35. It plays crucial roles in neuronal differentiation, cell proliferation, and DNA repair mechanisms . The protein is widely expressed across human tissues including brain, heart, placenta, liver, skeletal muscle, lung, kidney, and pancreas, with notable overexpression observed in certain tumor tissues . As a regulatory component of the G2/M DNA damage checkpoint responding to genotoxic stress, CDK5RAP3 is essential for maintaining genomic integrity . Recent research has revealed its function as a tumor suppressor in gastric cancer through inhibition of NF-κB nuclear transcription, which reduces cytokine secretion (specifically IL4 and IL10) and blocks M2 macrophage polarization . This immunomodulatory function makes CDK5RAP3 a potential target for cancer immunotherapy strategies.

What applications are CDK5RAP3 antibodies suitable for?

CDK5RAP3 antibodies have been validated for multiple experimental applications:

For optimal results, it is recommended to titrate the antibody concentration in each specific testing system as sensitivity may be sample-dependent . Antigen retrieval methods can significantly impact IHC results, with recommendations including TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0) .

How should CDK5RAP3 antibodies be stored and handled?

Most CDK5RAP3 antibodies should be stored at -20°C where they remain stable for one year after shipment . The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, although some preparations (particularly those in 20μL sizes) may contain 0.1% BSA as a stabilizer . When working with these antibodies, avoid repeated freeze-thaw cycles that can compromise antibody integrity and performance . Before use, allow the antibody to equilibrate to room temperature and gently mix by inversion rather than vortexing to prevent protein denaturation.

How can I verify CDK5RAP3 antibody specificity in my experimental system?

Verifying antibody specificity is critical for reliable results. For CDK5RAP3 antibodies, consider implementing these validation approaches:

• Knockout/Knockdown verification: Use CDK5RAP3 knockdown or knockout cell lines as negative controls to confirm signal specificity . Published studies have utilized this approach to validate antibody specificity.

• Peptide competition assay: Employ specific blocking peptides like the recombinant protein antigen with N-terminal His6-ABP tag corresponding to human CDK5RAP3 . Pre-incubate your primary antibody with excess blocking peptide before application to your samples. Genuine CDK5RAP3 signals should disappear or significantly diminish.

• Molecular weight verification: CDK5RAP3 has a calculated molecular weight of 57 kDa but is typically observed at approximately 65 kDa on Western blots . This discrepancy is due to post-translational modifications and should be consistently observed.

• Multiple antibody verification: Compare results using antibodies targeting different epitopes of CDK5RAP3. For instance, compare antibodies targeting N-terminal regions with those recognizing internal domains (amino acids 207-506) .

• Multiple detection methods: Confirm findings across different applications (e.g., WB, IHC, IF) to ensure consistency of results across methodologies.

What are the known challenges in detecting CDK5RAP3 isoforms?

CDK5RAP3 exists in three named isoforms resulting from alternative splicing , which presents specific detection challenges:

• Isoform-specific epitope availability: Depending on the antibody's target epitope, certain isoforms may not be detected. For comprehensive isoform detection, select antibodies targeting conserved regions or use multiple antibodies targeting different domains.

• Molecular weight discrepancies: Though calculated at 57 kDa, CDK5RAP3 typically appears at 65 kDa on Western blots due to post-translational modifications . These modifications may vary between tissue types and cellular conditions, potentially yielding multiple bands.

• Subcellular localization differences: CDK5RAP3 is broadly located in subcellular compartments . Different isoforms may preferentially localize to specific compartments, requiring appropriate subcellular fractionation protocols for comprehensive detection.

• Variation in expression levels: Expression levels of specific isoforms may vary substantially between tissues and experimental conditions. This might necessitate adjusting sample loading amounts or detection sensitivity parameters.

• Potential cross-reactivity: CDK5RAP3 contains structural motifs including two leucine zipper domains and putative phosphorylation sites that share homology with other proteins. Validate antibody specificity carefully to avoid false positives.

How does CDK5RAP3 function in the tumor microenvironment affect experimental design?

Recent research has revealed CDK5RAP3's role in regulating tumor-associated macrophages (TAMs) and the tumor microenvironment (TME) , which requires special experimental considerations:

• Co-culture systems: To study CDK5RAP3's effects on macrophage polarization, design co-culture experiments with gastric cancer cells (with and without CDK5RAP3 expression) and macrophages. Monitor macrophage phenotype shifts (M1/M2) using appropriate markers.

• Cytokine profiling: CDK5RAP3 inhibits NF-κB nuclear transcription, reducing IL4 and IL10 secretion . Include comprehensive cytokine profiling (ELISA or multiplex assays) in experimental designs studying CDK5RAP3's immunomodulatory functions.

• MMP2 activity assessment: The absence of CDK5RAP3 in gastric cancer cells induces macrophages to secrete more MMP2, promoting EMT processes . Include zymography or other MMP activity assays when studying invasion and migration in CDK5RAP3-deficient systems.

• In vivo tumor models: When designing in vivo experiments, consider analyzing TAM infiltration and polarization in tumors with varying CDK5RAP3 expression levels. Flow cytometry and immunohistochemistry with macrophage subset markers are essential components of such studies.

• EMT marker assessment: Given CDK5RAP3's impact on the EMT process through MMP2 regulation , incorporate analysis of EMT markers (E-cadherin, N-cadherin, vimentin) in experimental protocols.

What are the optimal conditions for Western blot detection of CDK5RAP3?

Achieving optimal Western blot results for CDK5RAP3 requires attention to several technical parameters:

• Sample preparation: Use whole cell lysates from appropriate cell lines with confirmed CDK5RAP3 expression. Validated cell lines include HEK-293T, HeLa, U-87 MG, A-431, and Jurkat cells .

• Antibody dilution: For primary antibody incubation, use a dilution range of 1:2000-1:12000 for polyclonal antibodies or 1:1000 for monoclonal antibodies like EPR11293 . For secondary detection, HRP-labeled anti-rabbit or anti-mouse antibodies at 1:2000 dilution have been successfully employed .

• Sample loading: Load approximately 10-20 μg of total protein per lane . Excessive protein loading may cause high background, while insufficient loading may result in weak or undetectable signals.

• Band identification: Expect to observe a band at approximately 65 kDa, despite the calculated molecular weight of 57 kDa . This discrepancy is due to post-translational modifications.

• Controls: Include positive control lysates (e.g., HEK-293T or HeLa cells) and consider using CDK5RAP3 knockdown/knockout samples as negative controls to confirm signal specificity .

What are the critical factors for successful immunohistochemistry and immunofluorescence with CDK5RAP3 antibodies?

For optimal IHC and IF results with CDK5RAP3 antibodies, consider these methodological factors:

• Antigen retrieval: For formalin-fixed, paraffin-embedded tissues, use TE buffer at pH 9.0 for antigen retrieval. Alternatively, citrate buffer at pH 6.0 may also be effective . The choice of antigen retrieval method significantly impacts staining sensitivity and specificity.

• Antibody dilution: Use a dilution range of 1:50-1:500 for both IHC and IF applications . The optimal dilution may vary depending on tissue type and fixation methods.

• Incubation conditions: For primary antibody incubation, overnight incubation at 4°C generally yields optimal results with minimal background. For IF applications, a 1:50 dilution has been validated in U87-MG cells .

• Signal detection systems: For IHC, both DAB-based and fluorescence-based detection systems are compatible. For IF, various secondary antibodies are available, including multiple Alexa Fluor® conjugates .

• Validated tissue types: Human stomach and liver tissues have been validated for IHC and IF applications . For other tissue types, additional optimization may be necessary.

How can I resolve weak or absent signals in CDK5RAP3 detection?

When encountering weak or absent signals when detecting CDK5RAP3, consider these troubleshooting approaches:

• Expression level verification: Confirm that your experimental system expresses CDK5RAP3. While widely expressed in human tissues, expression levels vary significantly. Consider using known positive controls such as HEK-293T, HeLa, or U-87 MG cells .

• Antibody concentration adjustment: If using dilutions at the higher end of the recommended range (e.g., 1:12000 for WB), try more concentrated antibody preparations (e.g., 1:2000) . Titrate to determine optimal concentration for your specific system.

• Antigen retrieval optimization: For IHC and IF applications, compare different antigen retrieval methods. While TE buffer (pH 9.0) is recommended, citrate buffer (pH 6.0) is an alternative that may yield better results in certain contexts .

• Sample preservation: Ensure proper sample handling and storage. Protein degradation can significantly impact detection. Use fresh samples or properly preserved specimens with appropriate protease inhibitors.

• Detection system sensitivity: Consider using more sensitive detection systems, such as enhanced chemiluminescence for Western blots or signal amplification systems for IHC/IF applications.

What strategies can address cross-reactivity or background issues with CDK5RAP3 antibodies?

High background or non-specific signals can compromise experimental results. Address these issues with these approaches:

• Blocking optimization: Increase blocking duration or test alternative blocking agents (BSA vs. non-fat dry milk vs. commercial blocking solutions). For polyclonal antibodies, matching the blocking agent to the host species of the secondary antibody can reduce background.

• Antibody selection: Consider switching from polyclonal to monoclonal antibodies like EPR11293 or B-11 for potentially improved specificity, particularly in applications prone to cross-reactivity.

• Antibody validation: Verify antibody specificity using appropriate controls, including CDK5RAP3 knockdown/knockout samples or peptide competition assays with recombinant protein antigens .

• Washing protocol optimization: Extend washing steps between antibody incubations and increase the number of washes to remove unbound or weakly bound antibodies.

• Species cross-reactivity consideration: While CDK5RAP3 antibodies are tested for reactivity with human samples, cited reactivity includes human, mouse, and bovine samples . When working with non-human samples, verify antibody cross-reactivity or select antibodies validated for your species of interest.

How can CDK5RAP3 antibodies be utilized in cancer research?

CDK5RAP3 has emerged as a significant tumor suppressor in gastric cancer , offering several research applications:

• Tumor suppressor pathway analysis: Use CDK5RAP3 antibodies to investigate its interaction with NF-κB and downstream effects on cytokine secretion (IL4/IL10) . Chromatin immunoprecipitation (ChIP) assays can elucidate the mechanism of transcriptional regulation.

• Tumor microenvironment studies: Employ CDK5RAP3 antibodies in immunohistochemistry to evaluate its expression in tumor tissues and correlate with macrophage infiltration and polarization . Multi-color immunofluorescence can simultaneously visualize CDK5RAP3 expression and macrophage phenotypes.

• Metastasis mechanism investigation: Given CDK5RAP3's role in inhibiting MMP2 secretion and EMT processes , use antibodies to track its expression during cancer progression and metastasis.

• Prognostic biomarker development: Analyze CDK5RAP3 expression in patient samples to evaluate its potential as a prognostic marker for gastric cancer and potentially other cancers.

• Therapeutic target validation: Use CDK5RAP3 antibodies to monitor protein expression and localization in response to experimental therapeutics aimed at restoring or enhancing its tumor suppressor function.

What considerations apply to using CDK5RAP3 antibodies in neuroscience research?

Given CDK5RAP3's initial identification as a binding protein of CDK5 activator p35 and its importance in neuronal differentiation , special considerations apply to neuroscience applications:

• Neuronal differentiation studies: Use CDK5RAP3 antibodies to monitor expression changes during neuronal differentiation processes. Immunofluorescence can visualize subcellular localization shifts during different developmental stages.

• CDK5 pathway investigation: Employ co-immunoprecipitation with CDK5RAP3 antibodies to study its interactions with CDK5 and p35 in neuronal systems. This can elucidate regulatory mechanisms in both normal development and pathological conditions.

• Brain tissue analysis: When using CDK5RAP3 antibodies in brain tissue analysis, consider region-specific expression patterns and potential variations in post-translational modifications that may affect antibody recognition.

• Neurodegenerative disease models: Investigate potential alterations in CDK5RAP3 expression or localization in models of neurodegenerative diseases where CDK5 dysregulation has been implicated.

• Primary neuronal culture applications: When working with primary neurons, optimize fixation and permeabilization protocols to preserve cellular architecture while ensuring antibody accessibility to CDK5RAP3 epitopes.

Comparative Analysis of CDK5RAP3 Antibody Properties

Recommended Dilutions for Different Applications