CRK12 Antibody

What is CRK12 and what are its primary functions?

CRK12 (Cdc2-related kinase, arginine/serine-rich) is a serine/threonine protein kinase that belongs to the CDK (Cyclin-Dependent Kinase) family. It functions primarily by forming complexes with specific cyclins, particularly CYC9. The CRK12:CYC9 complex forms an active protein kinase in organisms including trypanosomatid parasites, where it plays essential roles in cellular processes .

In Trypanosoma brucei, CRK12 has been demonstrated as essential for parasite proliferation and survival both in vitro and in mouse models, making it a validated drug target for trypanosomiasis . Functional characterization using RNA interference has revealed roles for CRK12 in critical cellular processes including endocytosis .

The human homolog CDK12 (also referred to as CRKRS) has a calculated molecular weight of 164 kDa (1490 amino acids) but is typically observed at 180-200 kDa in experimental conditions . The protein is encoded by the CDK12 gene (Gene ID: 51755) .

What are the key applications of CRK12 antibodies in research?

CRK12 antibodies serve multiple critical functions in research settings, with applications primarily in:

• Western Blot (WB): For detection and quantification of CRK12 protein in cell lysates

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualization of CRK12 localization within cells

• Immunoprecipitation (IP): For isolation of CRK12 and its binding partners

• ELISA: For quantitative detection of CRK12 in solution

These applications have been validated in published literature, with at least 7 publications utilizing CRK12 antibodies for Western blot and 1 publication each for immunoprecipitation and knockdown/knockout studies .

How should researchers select an appropriate CRK12 antibody?

When selecting a CRK12 antibody, researchers should consider:

• Reactivity: Confirm the antibody reacts with your species of interest. For example, the 26816-1-AP antibody has confirmed reactivity with human samples and predicted reactivity with other species .

• Application compatibility: Verify the antibody is validated for your intended application. Different applications require different antibody characteristics.

• Type of antibody: Consider whether monoclonal or polyclonal antibodies better suit your research. Polyclonal antibodies like 26816-1-AP recognize multiple epitopes, potentially providing stronger signals but possibly with more background .

• Validation data: Review existing validation data including Western blot images, immunofluorescence data, and published literature citations.

• Molecular weight confirmation: Ensure the antibody detects CRK12 at the expected molecular weight (180-200 kDa for human CRK12) .

What are the recommended dilutions for CRK12 antibodies in different applications?

Based on validated experimental protocols, the following dilutions are recommended for CRK12 antibodies:

It is strongly recommended that researchers titrate the antibody in each testing system to obtain optimal results, as required dilutions may be sample-dependent .

What cell lines have been validated for CRK12 antibody detection?

CRK12 antibodies have been validated in the following cell lines:

When working with other cell lines or tissue samples, preliminary validation experiments are recommended to confirm antibody specificity and optimal working conditions .

What are the proper storage and handling procedures for CRK12 antibodies?

For optimal performance and longevity of CRK12 antibodies:

• Storage temperature: Store at -20°C. Antibodies are typically stable for one year after shipment when stored properly .

• Buffer conditions: CRK12 antibodies are commonly provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: For the -20°C storage, aliquoting is generally unnecessary for small volume antibodies (20μl sizes) .

• Freeze-thaw cycles: Minimize freeze-thaw cycles to preserve antibody activity.

• Working dilutions: Prepare fresh working dilutions on the day of the experiment.

• BSA content: Note that some preparations (20μl sizes) may contain 0.1% BSA .

How does CRK12 interact with cyclins and what is the significance of the CRK12:CYC9 complex?

The CRK12:CYC9 interaction represents an important protein kinase complex with essential functions in trypanosomatids:

• Complex formation: CRK12 interacts with CYC9 (a putative transcriptional cyclin) to form an active protein kinase complex in both procyclic and bloodstream forms of Trypanosoma brucei .

• Essential nature: Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro. CRK12 has also been shown to be essential for survival of T. brucei in mouse models .

• Functional roles: Functional characterization using RNA interference has revealed distinct roles for these proteins:

  • CRK12: Involved in endocytosis

  • CYC9: Functions in cytokinesis

• Drug target validation: The essential nature of the CRK12:CYC9 complex provides genetic validation of this complex as a novel drug target for trypanosomiasis .

This complex differs from other CRK:cyclin interactions in trypanosomatids. For instance, CRK3 has been shown to bind to CYC2 and CYC6 in vivo, regulating G1/S and G2/M transitions .

How can researchers study CRK12 kinase activity?

To study CRK12 kinase activity, researchers can employ several methodological approaches:

• Expression of tagged proteins: Generate tetracycline-inducible cell lines expressing TY-tagged CRK12 (both active and kinase-dead variants) to investigate kinase activity. The kinase-dead variant can be created through site-directed mutagenesis (e.g., K358M mutation) .

• Kinase assays: Conduct in vitro kinase assays using immunoprecipitated CRK12 complexes to measure phosphorylation of substrate proteins.

• Inhibitor studies: Utilize CRK12 inhibitors such as CRK12-IN-2 to assess the effects of CRK12 inhibition on cellular processes and validate kinase activity .

• Genetic approaches: Implement RNA interference targeting CRK12 to study the phenotypic effects of CRK12 depletion .

For protein expression studies, researchers have successfully employed the following approaches:

• PCR amplification of the CRK12 ORF from genomic DNA

• Cloning into appropriate expression vectors with tags (GFP, TY)

• Creation of kinase-dead variants through site-directed mutagenesis

• Transfection into appropriate cell lines

• Confirmation of expression by Western blotting

What is known about CRK12 as a drug target in parasitic diseases?

CRK12 has emerged as a promising drug target for several parasitic diseases:

• Genetic validation: CRK12 has been shown to be essential for the survival of Trypanosoma brucei both in vitro and in mouse models, providing genetic validation as a drug target .

• Inhibitor development: Compounds such as CRK12-IN-2 (compound 2) have been developed as potent inhibitors of CRK12 .

• Species specificity: CRK12-IN-2 shows remarkable potency against trypanosomatid parasites:

  • Trypanosoma congolense: EC₅₀ value of 3.2 nM

  • Trypanosoma vivax: EC₅₀ value of 0.08 nM

• In vivo efficacy: CRK12-IN-2 (10 mg/kg, subcutaneous injection, once daily for 4 days) effectively cures mice with T. congolense and T. vivax infections .

• Computational approaches: Researchers have employed computational methods to model the 3D structure of Leishmania donovani CRK12 (LdCRK12) and screen for potential inhibitors. Natural product-derived compounds have been identified with predicted inhibitory constants (Ki) ranging from 0.108 to 0.587 μM .

• Binding mechanisms: Most CRK12 inhibitors bind in the ATP binding pocket of the kinase domain, with Lys488 identified as a key residue critical for ligand binding in the ATP binding pocket of LdCRK12 .

How can researchers optimize Western blot protocols for CRK12 detection?

For optimal Western blot detection of CRK12:

• Sample preparation:

  • Use appropriate lysis buffers that preserve protein integrity

  • Include protease and phosphatase inhibitors

  • Maintain samples at 4°C during processing

• Gel selection:

  • Use lower percentage gels (6-8%) to better resolve high molecular weight proteins like CRK12 (observed at 180-200 kDa)

• Transfer conditions:

  • For high molecular weight proteins, use longer transfer times or lower current

  • Consider semi-dry versus wet transfer based on protein size

• Antibody dilution optimization:

  • Begin with the recommended dilution range (1:2000-1:16000)

  • Perform a dilution series to determine optimal concentration

  • Consider longer incubation times at 4°C

• Detection method:

  • For low abundance proteins, consider more sensitive detection methods

  • Use appropriate exposure times to avoid oversaturation

• Positive controls:

  • Include lysates from cells known to express CRK12 (HeLa, K-562, or Jurkat cells)

• Troubleshooting non-specific bands:

  • Increase blocking time or blocking agent concentration

  • Adjust antibody dilution

  • Include additional washing steps

What methods can be used to validate CRK12 antibody specificity?

To ensure antibody specificity for CRK12:

• Genetic validation:

  • Use CRK12 knockdown/knockout samples as negative controls

  • Overexpression of tagged CRK12 as positive controls

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide

  • Specificity is indicated by signal reduction or elimination

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of CRK12

  • Concordant results suggest specific detection

• Mass spectrometry validation:

  • Immunoprecipitate with anti-CRK12 antibody

  • Confirm protein identity by mass spectrometry

• Cross-reactivity testing:

  • Test antibody against related kinases

  • Ensure specificity for CRK12 versus other CRKs

• Cell type specificity:

  • Compare detection in cells known to express CRK12 (HeLa, K-562, Jurkat) versus cells with low/no expression

How can researchers effectively use CRK12 antibodies for immunofluorescence studies?

For successful immunofluorescence detection of CRK12:

• Cell preparation:

  • Begin with validated cell lines such as A431 cells

  • Consider fixation method (paraformaldehyde vs. methanol)

  • Optimize permeabilization conditions

• Blocking conditions:

  • Use sufficient blocking to reduce background

  • Include serum from the same species as the secondary antibody

• Antibody dilution:

  • Start with recommended dilution range (1:300-1:1200)

  • Optimize through titration experiments

• Controls:

  • Include secondary-only controls to assess background

  • Use cells with CRK12 knockdown as negative controls

• Counterstaining:

  • Include nuclear stains (DAPI, Hoechst)

  • Consider co-staining with markers of relevant cellular compartments

• Imaging considerations:

  • Use appropriate exposure settings

  • Capture z-stacks for proper localization

  • Perform quantitative analysis if needed

• Troubleshooting:

  • For high background: increase blocking, reduce antibody concentration

  • For weak signal: increase antibody concentration, optimize fixation

What computational approaches are advancing CRK12 inhibitor discovery?

Computational methods are increasingly important in CRK12 inhibitor discovery:

• Structural modeling:

  • 3D molecular structure modeling of CRK12 from different species

  • Example: Leishmania donovani CRK12 (LdCRK12) has been modeled for virtual screening

• Virtual screening:

  • Screening of compound libraries against modeled CRK12 structures

  • Natural product libraries have been screened against LdCRK12

• Binding affinity prediction:

  • Prediction of inhibitory constants (Ki) for potential inhibitors

  • Compounds with Ki values ranging from 0.108 to 0.587 μM have been identified

• Molecular dynamics simulations:

  • Assessing stability of CRK12-inhibitor complexes

  • Molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) computations to reinforce binding predictions

• Binding site identification:

  • Identification of key residues in the ATP binding pocket

  • Lys488 has been identified as critical for ligand binding in LdCRK12

These computational approaches accelerate the discovery of novel CRK12 inhibitors, particularly from natural product sources, while reducing the need for initial high-throughput screening.

How does CRK12 inhibition affect different parasite species?

CRK12 inhibition shows varying effects across parasite species:

• Trypanosoma brucei:

  • CRK12 is essential for proliferation and survival

  • RNAi knockdown affects endocytosis

• Trypanosoma congolense:

  • Highly sensitive to CRK12-IN-2 with EC₅₀ of 3.2 nM

  • Mice with T. congolense infection can be fully cured with CRK12-IN-2 (10 mg/kg, s.c. once daily for 4 days)

• Trypanosoma vivax:

  • Extremely sensitive to CRK12-IN-2 with EC₅₀ of 0.08 nM

  • Complete cure in infected mice with CRK12-IN-2 treatment

• Leishmania donovani:

  • CRK12 identified as a plausible drug target

  • Computational studies have identified potential inhibitors for LdCRK12

These species-specific responses highlight the potential of CRK12 as a broad-spectrum target for antiparasitic drug development, particularly important as resistance to existing treatments increases.

What techniques are available for generating and validating CRK12 genetic models?

Researchers have several approaches for creating and validating CRK12 genetic models:

• Endogenous tagging:

  • PCR amplification of CRK12 ORF with appropriate restriction sites

  • Cloning into vectors enabling expression of tagged proteins (GFP, TY)

  • Linearization and transfection into target cell lines

  • Confirmation by Western blotting with anti-tag antibodies

• Inducible expression systems:

  • Generation of tetracycline-inducible cell lines

  • Expression of wild-type or mutant (kinase-dead) variants

  • Controlled expression for functional studies

• RNA interference (RNAi):

  • Design of RNAi constructs targeting specific CRK12 regions

  • Creation of stem-loop constructs for effective knockdown

  • Transfection and selection of stable cell lines

• Knockout approaches:

  • Generation of resistance-marker replacement constructs

  • Targeting 5' and 3' flanking regions of CRK12

  • Creation of single and double knockout lines when feasible

• Validation techniques:

  • Western blotting to confirm protein expression/depletion

  • Phenotypic analysis (growth curves, microscopy)

  • Functional assays specific to CRK12 roles (endocytosis, cytokinesis)