VRK2 Antibody, HRP conjugated

Basic Research Applications and Characterization

• What is VRK2 and why is it significant for research applications?

  Vaccinia-related kinase 2 (VRK2) is a serine/threonine kinase that plays significant roles in various cellular processes including cell survival, proliferation, and DNA damage response. With a calculated molecular weight of 58 kDa (508 amino acids) and observed molecular weight of approximately 50 kDa, VRK2 is encoded by the VRK2 gene (NCBI Gene ID: 7444) . The protein's importance in research stems from its involvement in several critical cellular pathways and its potential implications in neurodegenerative disorders, particularly those involving polyglutamine (polyQ) protein aggregation such as Huntington's disease . Understanding VRK2 function has become increasingly important as it has been identified as a risk factor in genome-wide association studies for certain neurological conditions .

• What is the difference between HRP-conjugated and unconjugated VRK2 antibodies?

  HRP-conjugated VRK2 antibodies have horseradish peroxidase directly attached to the antibody molecule, enabling direct detection through enzymatic colorimetric or chemiluminescent reactions without the need for secondary antibodies. In contrast, unconjugated VRK2 antibodies (like the 12946-1-AP) require a secondary antibody system for detection . The conjugation affects several experimental parameters:

  Feature	HRP-conjugated VRK2 Antibody	Unconjugated VRK2 Antibody
  Detection	Direct (one-step)	Indirect (requires secondary antibody)
  Protocol time	Shorter	Longer
  Signal amplification	Limited to single HRP molecule per antibody	Potentially higher (multiple secondary antibodies)
  Background	Potentially lower (fewer steps)	May be higher (more steps, cross-reactivity)
  Storage conditions	More sensitive to storage conditions	Generally more stable

  For experimental design purposes, conjugation status should be selected based on assay sensitivity requirements and available detection systems.

• What applications are validated for VRK2 antibodies in research settings?

  VRK2 antibodies have been validated for multiple research applications with specific dilution recommendations. Based on established protocols, applications include:

  Application	Validated Dilution Range	Comments
  Western Blot (WB)	1:500-1:2000	Multiple publications confirm efficacy
  Immunoprecipitation (IP)	0.5-4.0 μg per 1-3 mg lysate	Successfully detected in K-562 cells
  Immunohistochemistry (IHC)	1:50-1:500	Validated in human stomach tissue
  ELISA	Application-specific	Used in sandwich enzyme immunoassay formats

  For HRP-conjugated variants specifically, these dilutions may require optimization as the detection system differs from traditional two-step methods. It is recommended to titrate the antibody in each specific experimental system to determine optimal working concentrations .

• What sample types and species reactivity have been confirmed for VRK2 antibodies?

  VRK2 antibodies demonstrate reactivity with samples from multiple species and diverse tissue/cell types:

  Species Reactivity	Confirmed Sample Types
  Human	HepG2 cells, liver tissue, stomach tissue, K-562 cells, U2OS cells, BxPC-3 cells
  Mouse	Skeletal muscle tissue
  Rat	Tested positive (specific tissues not detailed)

  For experimental applications using HRP-conjugated VRK2 antibodies, these validated sample types provide a starting point for experimental design. The antibody has been successfully employed in tissue homogenates, cell lysates, and other biological fluids .

Advanced Research Applications

• How does VRK2 influence polyglutamine protein aggregation in neurodegenerative disease models?

  VRK2 plays a critical role in polyglutamine (polyQ) protein aggregation through its regulatory effect on the eukaryotic chaperonin TRiC hetero-oligomeric complex. Research demonstrates that VRK2 functions as a negative regulator of TRiC by promoting its ubiquitination and subsequent degradation .

  The mechanism involves several steps:

  1. VRK2 directly interacts with specific subunits of the TRiC complex (particularly CCT1 and CCT4)

  2. This interaction leads to recruitment of COP1 E3 ligase complex components, notably through VRK2's binding with RBX1

  3. The activated E3 ligase complex promotes TRiC ubiquitination

  4. Ubiquitinated TRiC undergoes proteasomal degradation

  5. Reduced TRiC levels diminish cellular capacity to properly fold polyQ proteins

  6. Consequently, polyQ-expanded proteins (like mutant huntingtin) form toxic aggregates

  Experimental evidence shows that overexpression of wild-type VRK2 increases polyQ aggregation approximately 3-fold compared to controls, while kinase-dead VRK2 mutants show no such effect. Conversely, siRNA-mediated knockdown of VRK2 decreases polyQ aggregation by enhancing TRiC stability .

• What are the protein-protein interaction networks of VRK2 relevant to experimental design?

  When designing experiments using VRK2 antibodies, it's important to consider the protein's known interactions, particularly:

  Interaction Partner	Nature of Interaction	Functional Significance
  TRiC complex	Direct binding to CCT1/CCT4 subunits	Regulates TRiC stability and protein folding
  RBX1	Component of COP1 E3 ligase complex	Mediates ubiquitination of target proteins
  COP1 E3 ligase	Recruited by VRK2	Executes ubiquitination of TRiC

  Immunoprecipitation experiments have confirmed these interactions, showing that VRK2 efficiently co-immunoprecipitates with HA-tagged CCT1 or CCT4 when expressed in HEK293T cells . For co-immunoprecipitation studies using VRK2 antibodies, these established interactions provide positive controls and experimental validation markers.

• How can VRK2 antibodies be used to investigate neurodegeneration mechanisms?

  VRK2 antibodies provide valuable tools for investigating neurodegeneration through several methodological approaches:

  1. VRK2 expression profiling: Western blotting with VRK2 antibodies can assess protein levels in patient samples or disease models. Research suggests VRK2 levels may be critical for the onset and progression of polyQ diseases, making quantitative analysis valuable .

  2. Co-localization studies: Immunofluorescence using VRK2 antibodies alongside markers for protein aggregation (like mutant huntingtin) can reveal spatial relationships between VRK2 expression and aggregate formation.

  3. TRiC-VRK2 interaction analysis: Co-immunoprecipitation with VRK2 antibodies can pull down TRiC complex components, allowing assessment of their interaction status in disease states.

  4. Phosphorylation studies: Given VRK2's role as a serine/threonine kinase, phospho-specific antibodies can help identify substrates and signaling pathways altered in neurodegenerative conditions.

  5. Therapeutic target validation: In knockdown/overexpression studies, VRK2 antibodies can confirm modulation of protein levels when testing potential interventions targeting the VRK2-TRiC pathway.

  These applications are particularly relevant as studies have demonstrated that VRK2 overexpression increases polyQ aggregation, while its knockdown has a protective effect against aggregation .

• What are the structural and functional differences between VRK2 isoforms relevant to antibody selection?

  When selecting VRK2 antibodies for experiments, researchers should consider the existence of multiple VRK2 isoforms that may impact antibody binding and experimental interpretation:

  The VRK2 gene produces at least two main isoforms with distinct subcellular localizations and potentially different functions. Antibody epitope location determines whether specific isoforms or all isoforms will be detected. The VRK2 antibody (12946-1-AP) was generated using a fusion protein immunogen (Ag4019) , and researchers should verify which isoforms this antibody recognizes for their specific application.

  Fluorescent imaging analysis has shown VRK2 localizes to the endoplasmic reticulum, mitochondria, and nuclear envelope, likely due to its transmembrane domain . For immunofluorescence applications, this localization pattern can serve as a validation marker for antibody specificity.

Methodological Considerations

• What are the optimal protocol conditions for Western blotting with VRK2 antibodies?

  For optimal Western blot results with VRK2 antibodies, the following methodological parameters are recommended:

  Parameter	Recommendation	Notes
  Dilution	1:500-1:2000	For unconjugated; HRP-conjugated may require different dilution
  Sample loading	20-40 μg total protein	For cell/tissue lysates
  Expected band size	~50 kDa	Observed molecular weight; calculated MW is 58 kDa
  Transfer method	Wet transfer recommended	For optimal transfer of proteins >40 kDa
  Blocking solution	5% non-fat milk or BSA in TBST	1 hour at room temperature
  Primary antibody incubation	Overnight at 4°C	For unconjugated antibodies
  Detection	Standard ECL	Adjust exposure time based on signal intensity

  For HRP-conjugated VRK2 antibodies specifically, eliminate the secondary antibody step and proceed directly to detection after washing. Positive controls from validated cell lines include HepG2, K-562, U2OS, and BxPC-3 cells .

• What antigen retrieval methods are most effective for immunohistochemistry with VRK2 antibodies?

  For successful immunohistochemical detection of VRK2, appropriate antigen retrieval is critical:

  Recommended Method	Alternative Method	Dilution Range
  TE buffer pH 9.0	Citrate buffer pH 6.0	1:50-1:500

  The effectiveness of antigen retrieval depends on tissue fixation procedures, embedding methods, and section thickness. For formalin-fixed paraffin-embedded tissues, heat-induced epitope retrieval (HIER) with TE buffer at pH 9.0 has shown optimal results for VRK2 detection in human stomach tissue . Incubation times for primary antibody typically range from 1-2 hours at room temperature or overnight at 4°C, with subsequent detection using an appropriate HRP-conjugated secondary antibody system (or direct detection if using HRP-conjugated primary antibodies).

• What positive and negative controls should be included when validating VRK2 antibody specificity?

  Proper experimental controls are essential for validating VRK2 antibody specificity:

  Positive Controls:

  • Cell lines: HepG2, K-562, U2OS, and BxPC-3 cells (validated for Western blot)

  • Tissues: Human liver, mouse skeletal muscle, human stomach (validated for WB and IHC)

  • Overexpression systems: HEK293T cells transiently transfected with VRK2 expression vectors

  Negative Controls:

  • Antibody controls: Isotype control (rabbit IgG for 12946-1-AP)

  • Sample controls: VRK2 knockdown cells using validated siRNA (confirms signal specificity)

  • Peptide competition assay: Pre-incubation of antibody with immunogen peptide should abolish specific signal

  For knockout validation, the literature includes at least two publications using knockout/knockdown approaches that can serve as reference points for expected results .

• How can immunoprecipitation with VRK2 antibodies be optimized for protein interaction studies?

  For successful immunoprecipitation of VRK2 and its interacting partners:

  Parameter	Recommendation	Notes
  Antibody amount	0.5-4.0 μg	Per 1.0-3.0 mg total protein lysate
  Lysis buffer	RIPA or NP-40 based	Include protease and phosphatase inhibitors
  Pre-clearing	1 hour	With protein A/G beads to reduce background
  Antibody binding	Overnight at 4°C	With gentle rotation
  Washing	4-5 times	Increasingly stringent washes

  When studying VRK2-TRiC interactions specifically, published protocols have successfully demonstrated co-immunoprecipitation of VRK2 with CCT subunits (1, 2, 4, and 7) in A549 cells, which express high levels of endogenous VRK2 . For protein interaction studies, gentler lysis conditions may better preserve protein-protein interactions compared to RIPA buffer.

Troubleshooting and Advanced Applications

• What approaches can resolve common issues with HRP-conjugated antibody detection systems?

  When troubleshooting HRP-conjugated VRK2 antibody experiments:

  Issue	Possible Cause	Solution
  High background	Excessive antibody concentration	Titrate antibody to optimal dilution
  Weak signal	Inadequate antigen, inactive HRP	Increase protein loading, verify HRP activity
  Non-specific bands	Cross-reactivity, protein degradation	Use more stringent washing, add protease inhibitors
  Signal variability	Storage degradation of conjugate	Aliquot antibody, store at -20°C with glycerol

  For optimal performance, HRP-conjugated antibodies should be stored at -20°C with 50% glycerol and 0.02% sodium azide at pH 7.3, similar to the storage conditions recommended for unconjugated VRK2 antibodies . Antibodies are generally stable for one year after shipment under these conditions, and small (20μl) sizes may contain 0.1% BSA as stabilizer .

• How can VRK2 antibodies be used to study polyglutamine aggregation mechanisms?

  VRK2 antibodies provide valuable tools for investigating polyglutamine aggregation through several experimental approaches:

  1. Co-localization studies: Immunofluorescence with VRK2 antibodies alongside fluorescently-tagged polyQ proteins can determine spatial relationships. Research has shown that overexpression of wild-type VRK2 increases HttQ103-GFP aggregate formation approximately 3-fold over control conditions .

  2. Biochemical assessment: Filter-trap assays and Western blotting of SDS-resistant aggregates can quantify aggregation levels in VRK2-manipulated systems. Published studies show that VRK2 overexpression increases SDS-resistant aggregates detectable by filter-trap assay .

  3. Rescue experiments: VRK2-mediated increase in polyQ aggregation can be mitigated by CCT4 co-expression, suggesting the VRK2-TRiC regulatory axis as a potential therapeutic target .

  4. Knockdown validation: siRNA-mediated VRK2 knockdown decreases polyQ aggregation and enhances TRiC protein stability, providing mechanistic insight into VRK2's role .

  These approaches have revealed that VRK2's kinase activity is essential for its effects on polyQ aggregation, as kinase-dead VRK2 mutants do not promote HttQ103-GFP aggregate formation .

• What methodological considerations are important when using VRK2 antibodies for ELISA applications?

  When implementing VRK2 antibodies in ELISA applications:

  1. Sandwich ELISA design: For detecting native VRK2, sandwich ELISA formats using capture and detection antibodies are recommended. The detection system typically employs biotin-conjugated antibodies and Avidin-HRP conjugates .

  2. Standards preparation: Serial dilutions of recombinant VRK2 protein standards should be prepared to generate a standard curve spanning the expected concentration range in experimental samples.

  3. Sample preparation: For tissue homogenates and cell lysates, gentle lysis buffers that maintain protein conformation are preferred over harsh detergents .

  4. Validation: ELISA specificity should be validated against both positive controls (samples known to express VRK2) and negative controls (samples with VRK2 knockdown).

  5. Data analysis: After TMB substrate addition and sulfuric acid reaction termination, absorbance readings at 450nm ± 10nm are used to calculate VRK2 concentrations by comparison to the standard curve .

  Commercial VRK2 ELISA kits employ pre-coated microwell plates with VRK2-specific antibodies and have been validated for detecting native (not recombinant) VRK2 in various biological samples .

• How can VRK2 antibody applications be integrated with functional studies of protein degradation pathways?

  VRK2 antibodies can be powerfully integrated with ubiquitin-proteasome pathway studies:

  1. Ubiquitination assays: VRK2 promotes TRiC ubiquitination, making ubiquitin co-immunoprecipitation an important application. Research demonstrates that wild-type VRK2 decreases endogenous TRiC levels by promoting ubiquitination, while kinase-dead VRK2 mutants do not .

  2. E3 ligase activity assessment: VRK2 recruits COP1 E3 ligase complex components and interacts specifically with RBX1, increasing E3 ligase activity on TRiC in vitro . Antibodies against VRK2 can help purify components for reconstituted ubiquitination assays.

  3. Proteasome inhibition studies: VRK2-mediated TRiC degradation occurs through the proteasomal pathway, which can be confirmed by treating cells with proteasome inhibitors while monitoring TRiC levels via Western blot .

  4. Pulse-chase experiments: Combining VRK2 antibodies with metabolic labeling can determine protein turnover rates of TRiC in the presence or absence of VRK2 activity.

  These approaches have established that VRK2 functions as a critical regulator of the ubiquitination-proteasomal degradation of TRiC, which in turn controls the folding of polyglutamine proteins involved in Huntington's disease .