CDK13 Antibody

What is CDK13 and why is it a significant research target?

CDK13 is a 164.9 kDa protein (1512 amino acids) that functions as a cyclin-dependent kinase with roles in transcriptional regulation and RNA processing. It belongs to the CMGC Ser/Thr protein kinase family and has nuclear subcellular localization . CDK13 is significant because it:

• Displays CTD kinase activity by hyperphosphorylating the C-terminal heptapeptide repeat domain of RNA polymerase II subunit RPB1

• Regulates RNA splicing, likely through phosphorylation of splicing factors like SRSF1/SF2

• Has tissue-specific expression patterns in brain, liver, muscle, and fetal tissues

• Forms functional complexes with cyclin K to regulate various cellular processes

• Is implicated in multiple cancer types, including hepatocellular carcinoma and colorectal cancer

Understanding CDK13 function has implications for both basic cell biology and potential therapeutic development for conditions where CDK13 regulation is disrupted.

What are the key considerations when selecting a CDK13 antibody for research?

When selecting a CDK13 antibody, researchers should consider:

• Target epitope location: CDK13 has multiple domains with the kinase domain (amino acids 604-1030) being critical for cyclin K interaction

• Antibody format: Both monoclonal (greater specificity) and polyclonal (broader epitope recognition) options are available from over 20 suppliers

• Validated applications: Choose antibodies specifically validated for your application (Western blot, IHC, IF, flow cytometry, etc.)

• Species reactivity: Ensure compatibility with your experimental model; human-reactive antibodies may not cross-react with rodent targets

• Clonality: Monoclonal antibodies like clone 46B7-G7 provide consistent lot-to-lot reproducibility for longitudinal studies

• Immunogen information: Some antibodies target specific regions, such as amino acids 1150-1300 or 1391-1415

Researchers should review validation data, including Western blot images showing the expected 164.9-192 kDa band, before selecting an antibody for their specific application.

How can I distinguish between CDK12 and CDK13 in my experiments?

Distinguishing between CDK12 and CDK13 requires careful antibody selection and experimental design due to their high sequence homology:

• Sequence similarity: CDK12 and CDK13 share >90% sequence identity in their kinase domains

• Antibody selection: Use antibodies raised against non-conserved regions, particularly in the N- or C-terminal domains where these proteins differ significantly

• Validation techniques:

  • Perform immunoprecipitation followed by mass spectrometry to confirm specificity

  • Use siRNA/shRNA knockdown controls specific to each protein

  • Include recombinant protein standards of both CDK12 and CDK13 in Western blots

• Western blot identification: CDK13 typically appears at ~192 kDa on Western blots, while CDK12 appears at a slightly different molecular weight

Mass spectrometry analysis has shown that many peptides are shared between these proteins, making definitive identification challenging. In one study, nine of ten unique peptides were found to be common to both kinases , highlighting the importance of using multiple validation approaches.

What are the optimal conditions for Western blot detection of CDK13?

For optimal Western blot detection of CDK13:

• Sample preparation:

  • Use nuclear extraction protocols to enrich for CDK13 (a nuclear protein)

  • Include phosphatase inhibitors to preserve phosphorylation status

  • Denature samples at 95°C for 5 minutes in Laemmli buffer with DTT

• Gel separation:

  • Use 6-8% SDS-PAGE gels due to CDK13's large size (164.9-192 kDa)

  • Run at lower voltage (80-100V) for better resolution of high molecular weight proteins

• Transfer conditions:

  • Perform wet transfer at 30V overnight at 4°C to ensure complete transfer of large proteins

  • Use PVDF membrane (0.45 μm pore size) rather than nitrocellulose

• Antibody incubation:

  • Block with 5% BSA in TBST (not milk, which can interfere with phospho-epitope detection)

  • Dilute primary antibodies at 1:1000 concentration

  • Incubate overnight at 4°C with gentle agitation

• Detection controls:

  • Include positive control lysates from cells known to express CDK13 (HEK293 cells work well)

  • Use molecular weight markers spanning 150-250 kDa range

Following these protocols should yield a clean band at approximately 192 kDa corresponding to CDK13 .

How can I optimize immunofluorescence staining for CDK13 in different cell types?

For successful immunofluorescence detection of CDK13:

• Fixation methods:

  • 4% paraformaldehyde (15 minutes at room temperature) preserves cellular architecture

  • For phospho-specific detection, include phosphatase inhibitors in fixation buffers

• Permeabilization:

  • Use 0.1% Triton X-100 for 10 minutes for adequate nuclear penetration

  • Alternative: 0.5% saponin works well for some antibody clones

• Blocking conditions:

  • 5% normal serum (matching secondary antibody host) with 0.1% BSA

  • 1-hour incubation at room temperature

• Antibody incubation:

  • Dilute primary antibodies according to validation data (typically 1:100-1:500)

  • Incubate overnight at 4°C in a humidified chamber

  • Rabbit polyclonal antibodies have shown good results for ICC/IF applications

• Nuclear counterstaining:

  • DAPI or Hoechst at appropriate dilutions

  • CDK13 should colocalize with nuclear staining due to its nuclear localization

• Mounting and imaging:

  • Use antifade mounting media to prevent photobleaching

  • Confocal microscopy is recommended to clearly visualize nuclear localization patterns

Cell-type specific considerations: Different cell lines may require optimization of antibody concentration due to varying expression levels of CDK13 across tissue types .

What approaches can be used to study CDK13-cyclin K interactions?

To investigate CDK13-cyclin K interactions, researchers can employ several complementary approaches:

• Co-immunoprecipitation:

  • Use anti-CDK13 antibodies to pull down protein complexes, then probe for cyclin K

  • The kinase domain of CDK13 (amino acids 604-1030) is sufficient for cyclin K binding

  • Include appropriate controls (IgG, lysate input)

• Bimolecular fluorescence complementation:

  • Tag CDK13 and cyclin K with complementary fragments of fluorescent proteins

  • Interaction brings fragments together, restoring fluorescence

• Proximity ligation assay:

  • Enables visualization of protein interactions in situ

  • Provides spatial information about where in the nucleus interactions occur

• Sequential affinity purification:

  • Tag CDK13 with epitopes like FLAG-His₆ for tandem purification

  • Analyze complex by silver staining and mass spectrometry

  • This approach has successfully identified cyclin K as a CDK13-interacting protein

Example purification protocol validated in research:

• Express FLAG-His₆-tagged CDK13 in cells

• Generate nuclear extracts

• Perform anti-FLAG immunoprecipitation

• Elute and purify using nickel-nitrilotriacetic acid

• Separate by SDS-PAGE and visualize by silver staining

• Confirm interactions by mass spectrometry and Western blotting

How can CDK13 phosphorylation status be accurately measured?

Measuring CDK13 phosphorylation presents technical challenges that can be addressed through several approaches:

• Phospho-specific antibodies:

  • Currently limited commercial availability for CDK13 phospho-sites

  • Researchers may need to generate custom antibodies against predicted sites

• Phos-tag™ SDS-PAGE:

  • Incorporate Phos-tag™ into acrylamide gels to retard phosphorylated proteins

  • Enables visualization of different phosphorylation states as mobility shifts

  • Use 6% acrylamide gels with 50 μM Phos-tag™ and 100 μM MnCl₂

• Mass spectrometry approaches:

  • Immunoprecipitate CDK13 using validated antibodies

  • Analyze by LC-MS/MS with phospho-enrichment

  • Phosphorylated peptides can be identified and quantified

• In vitro kinase assays:

  • Purify CDK13-cyclin K complexes

  • Incubate with ATP and potential substrates

  • Measure phosphorylation by autoradiography or phospho-specific antibodies

• Phosphatase controls:

  • Treat samples with lambda phosphatase to confirm phosphorylation-dependent signals

  • Include phosphatase inhibitors in parallel samples as controls

These techniques can help researchers determine both the sites and stoichiometry of CDK13 phosphorylation, providing insights into its regulation and activity.

What approaches are effective for studying CDK13's role in transcriptional regulation and RNA processing?

To investigate CDK13's function in transcriptional regulation and RNA processing:

• Chromatin immunoprecipitation (ChIP):

  • Use validated CDK13 antibodies to identify genomic binding sites

  • Combine with sequencing (ChIP-seq) for genome-wide binding profiles

  • Focus on genes involved in DNA damage, stress response, and heat shock pathways

• RNA-seq after CDK13 depletion:

  • Use siRNA or CRISPR-Cas9 to deplete CDK13

  • Analyze alternative splicing patterns and transcript levels

  • Compare with CDK12 depletion to identify unique and shared targets

• PRO-seq (Precision Run-On sequencing):

  • Measures nascent transcription to distinguish direct transcriptional effects

  • Can identify CDK13-dependent changes in transcription elongation rates

• Co-IP followed by RNA-seq:

  • Immunoprecipitate CDK13 protein complexes

  • Extract and sequence associated RNAs

  • Identifies RNA targets directly bound by CDK13 complexes

• CTD phosphorylation assays:

  • Measure CDK13's ability to phosphorylate the C-terminal domain of RNA Polymerase II

  • Use phospho-specific antibodies against different CTD phosphorylation sites (Ser2, Ser5, Ser7)

  • Compare with known CTD kinases (CDK9, CDK12) to identify CDK13-specific patterns

These approaches can help distinguish CDK13's direct effects on transcription from secondary effects on RNA processing.

What are the key considerations when using CDK13 antibodies in cancer research?

For cancer research applications involving CDK13 antibodies:

• Expression level analysis:

  • CDK13 shows aberrant expression in hepatocellular carcinoma, colorectal cancer, and MYC-dependent ovarian cancers

  • Use immunohistochemistry with proper positive and negative controls

  • Score staining intensity on standardized scales (0-3+)

• Patient sample considerations:

  • Use antigen retrieval techniques optimized for FFPE tissues

  • Include normal adjacent tissue controls

  • Consider tissue microarrays for high-throughput screening

• Correlation with clinical parameters:

  • Document CDK13 expression patterns relative to:

    • Tumor grade and stage

    • Patient outcome data

    • Treatment response

• Therapeutic targeting validation:

  • Verify antibody specificity before evaluating CDK13 inhibitor effects

  • Use multiple antibodies targeting different epitopes

  • Include genetic knockdown controls alongside pharmacological inhibition

Researchers should be aware that CDK13's role may differ across cancer types, necessitating careful validation in each specific cancer context.

How can I validate CDK13 antibody specificity for my experiments?

A comprehensive validation strategy for CDK13 antibodies should include:

• Genetic approaches:

  • siRNA/shRNA knockdown of CDK13 should reduce antibody signal

  • CRISPR-Cas9 knockout provides the strongest specificity control

  • Validated shRNA constructs (e.g., hK-1 and hK-2) targeting different regions of CDK13 have been documented

• Overexpression controls:

  • Express tagged CDK13 and confirm co-detection with anti-tag and anti-CDK13 antibodies

  • Include mutant versions lacking the antibody epitope as negative controls

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide before application

  • Should abolish specific signal, as demonstrated for cyclin K antibodies

• Recombinant protein standards:

  • Include purified CDK13 protein at known concentrations

  • Especially important when quantifying expression levels

• Cross-reactivity assessment:

  • Test against closely related proteins, particularly CDK12

  • Given the high homology in kinase domains (>90%), cross-reactivity is a significant concern

A well-validated antibody should show a single band at approximately 192 kDa in Western blot applications and minimal background staining in immunofluorescence or immunohistochemistry.

What are common pitfalls in CDK13 antibody-based experiments and how can they be avoided?

Common pitfalls and their solutions include:

• Incorrect band identification:

  • CDK13 appears at 164.9-192 kDa, significantly higher than its predicted weight due to post-translational modifications

  • Always use molecular weight markers and positive control lysates

• Cross-reactivity with CDK12:

  • Due to >90% sequence identity in kinase domains

  • Verify specificity using recombinant proteins or knockout controls

  • Use antibodies targeting non-conserved regions when possible

• Inconsistent nuclear signal in immunofluorescence:

  • Inadequate fixation or permeabilization can prevent nuclear antibody access

  • Optimize permeabilization with 0.1-0.5% Triton X-100

  • Include controls for nuclear envelope integrity

• Variable immunoprecipitation efficiency:

  • Some epitopes may be masked in protein complexes

  • Try multiple antibodies targeting different regions

  • Mild detergents may help expose epitopes without disrupting interactions

• Background in tissue staining:

  • Optimize blocking conditions (5% normal serum matching secondary antibody host)

  • Include absorption controls with recombinant CDK13

  • Use monoclonal antibodies when possible to reduce non-specific binding

Maintaining consistent experimental conditions, including cell culture conditions, lysis buffers, and antibody lots, can significantly improve reproducibility when working with CDK13 antibodies.

How should results be interpreted when contradictory data emerge from different CDK13 antibodies?

When faced with contradictory results from different CDK13 antibodies:

• Evaluate antibody characteristics:

  • Compare epitopes targeted by each antibody

  • Antibodies recognizing different domains may give different results if:

    • Protein interactions mask specific epitopes

    • Post-translational modifications affect epitope accessibility

    • Alternative splicing creates isoform-specific epitopes

• Perform comprehensive validation:

  • Test all antibodies against the same positive and negative controls

  • Include genetic knockout/knockdown controls for each antibody

  • Verify with orthogonal methods (e.g., mass spectrometry)

• Consider cellular context:

  • CDK13 forms complexes with cyclin K and other proteins

  • Different antibodies may have different access to epitopes depending on complex formation

  • Cell type-specific post-translational modifications may affect antibody binding

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes in parallel

  • Implement genetic tagging approaches (FLAG, HA) as independent detection methods

  • When possible, confirm protein identity by mass spectrometry after immunoprecipitation

  • Document all antibody information (catalog number, lot, dilution) for reproducibility

Scientists should remember that contradictory results often lead to new biological insights about protein conformation, modification state, or interaction partners, and should be investigated thoroughly rather than dismissed.

How can CDK13 antibodies be used to investigate its role in disease models beyond cancer?

CDK13 antibodies can be valuable tools for investigating its roles in various disease contexts:

• Neurodevelopmental disorders:

  • CDK13 is expressed in brain tissue and may affect neuronal development

  • Immunohistochemistry can map expression patterns across brain regions

  • Co-localization with neuronal markers can reveal cell type-specific expression

• Viral infections:

  • CDK13 interacts with HIV-1 Tat protein and affects viral mRNA splicing

  • Antibodies can be used to track CDK13-viral protein interactions

  • Phospho-specific antibodies may reveal infection-induced changes in CDK13 activity

• Developmental disorders:

  • Given CDK13's expression in fetal tissues , antibodies can track developmental expression patterns

  • Immunohistochemistry on developmental tissue arrays can map temporal expression

  • Co-staining with differentiation markers can reveal stage-specific functions

• Immune system regulation:

  • CDK13 has roles in the innate immune system

  • Flow cytometry with CDK13 antibodies can profile expression across immune cell populations

  • Phospho-specific antibodies may reveal activation-dependent changes in CDK13

Researchers should select antibodies validated in the specific model system and cell types relevant to their disease of interest.

What are the considerations for using CDK13 antibodies in combination with inhibitor studies?

When combining CDK13 antibodies with CDK inhibitor studies:

• Epitope accessibility concerns:

  • Inhibitor binding may alter protein conformation

  • Test whether antibody binding is affected by inhibitor presence in vitro

  • Use multiple antibodies recognizing different epitopes

• Monitoring inhibitor effects:

  • Phospho-specific antibodies can verify kinase inhibition

  • Total CDK13 antibodies can confirm that expression level changes aren't confounding inhibition results

  • Monitor effects on known CDK13 substrates (e.g., RNA Pol II CTD phosphorylation)

• Selectivity verification:

  • Many CDK inhibitors affect multiple CDK family members

  • Combine antibody-based detection of multiple CDKs to assess inhibitor specificity

  • Include genetic knockdown controls alongside pharmacological inhibition

• Temporal considerations:

  • Establish time course of inhibitor effects

  • Some effects may be direct (kinase inhibition) while others are indirect (transcriptional changes)

  • Acute vs. chronic inhibition may produce different cellular responses

• Resistance mechanisms:

  • Monitor for compensatory changes in CDK13 expression or phosphorylation

  • Screen for altered interactions with cyclin K or other partners

  • Look for changes in related CDKs (especially CDK12) that might compensate for CDK13 inhibition

These approaches can help distinguish between on-target and off-target effects of CDK inhibitors in research and drug development contexts.

How can new antibody technologies advance CDK13 research?

Emerging antibody technologies offer new possibilities for CDK13 research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows access to epitopes in crowded nuclear environments

  • Can be expressed intracellularly for live-cell imaging of CDK13

  • May recognize conformational epitopes not accessible to conventional antibodies

• BiTE (Bispecific T-cell Engager) technology:

  • For targeted degradation of CDK13 in specific cell populations

  • Alternative to genetic knockout for studying CDK13 function

  • Allows temporal control of CDK13 depletion

• Intrabodies with degrons:

  • Express anti-CDK13 antibody fragments fused to degrons intracellularly

  • Enables rapid protein degradation without genetic modification

  • Allows temporal control of CDK13 levels

• Multiplex imaging technologies:

  • Cyclic immunofluorescence or mass cytometry

  • Can simultaneously detect CDK13, cyclin K, and multiple downstream targets

  • Reveals cell-to-cell variability in CDK13 complex formation and activity

• Antibody-guided proximity labeling:

  • Fuse peroxidase to anti-CDK13 antibodies

  • Allows spatial mapping of the CDK13 interactome

  • Can reveal cell type-specific or condition-specific interaction partners

These technologies may help overcome current limitations in studying CDK13 function and regulation, particularly regarding its dynamic interactions in the nucleus and roles in transcriptional regulation.