ROCK2 Antibody

What is ROCK2 and why is it an important research target?

ROCK2 (Rho-associated coiled-coil containing protein kinase 2) is a 160.9 kilodalton intracellular enzyme that plays a crucial role in regulating the actin cytoskeleton, which is essential for various cellular processes including cell shape maintenance, motility, and division. ROCK2 is also known as ROCK-II, rho-associated protein kinase 2, and p164 ROCK-2 . Its activity is tightly regulated by Rho GTPases, which function as molecular switches controlling cytoskeletal dynamics . ROCK2 has gained significant research attention because its dysregulation has been implicated in several pathological conditions, particularly cancer metastasis and cardiovascular diseases, making it an important target for therapeutic intervention . Additionally, recent research has identified ROCK2 as a target of autoimmunity in paraneoplastic encephalitis associated with urogenital cancer, expanding its importance in immunological and neurological research contexts .

What are the key differences between polyclonal and monoclonal ROCK2 antibodies in experimental applications?

Polyclonal ROCK2 Antibodies:

• Recognize multiple epitopes on the ROCK2 protein, increasing detection sensitivity

• Typically derived from rabbit or goat immunization with ROCK2 peptides

• Advantageous for detecting low-abundance ROCK2 expression in tissues

• Examples include Bioss Inc. ROCK2 polyclonal antibody, effective for WB, ELISA, ICC, IF, IHC-fr, and IHC-p applications

• More tolerant to minor protein denaturation or conformational changes

Monoclonal ROCK2 Antibodies:

• Target a single specific epitope, providing greater specificity

• Often derived from mouse hybridomas, such as the D-11 clone (mouse IgG2a kappa light chain)

• Superior for distinguishing between ROCK1 and ROCK2 isoforms

• Example: BosterBio Anti-ROCK2 Monoclonal Antibody is optimized for WB, ICC, and IF techniques

• Provide more consistent results between experiments and antibody lots

Selection criteria should be based on the specific experimental design, with monoclonals preferred for isoform differentiation and polyclonals for maximizing detection sensitivity in complex tissue samples.

How can researchers validate ROCK2 antibody specificity for their experimental systems?

Methodological validation of ROCK2 antibody specificity requires a multi-step approach:

• Knockout/knockdown controls:

  • Generate ROCK2 knockout cell lines using CRISPR-Cas9 or siRNA knockdown

  • Compare antibody signal between wild-type and knockout/knockdown samples

  • Complete loss or significant reduction of signal confirms specificity

• Peptide competition assay:

  • Pre-incubate the ROCK2 antibody with excess recombinant ROCK2 protein or immunizing peptide

  • Perform side-by-side immunostaining or western blot with blocked and unblocked antibody

  • Specific antibodies will show diminished signal in the blocked condition, as demonstrated in competitive inhibition experiments

• Cross-reactivity assessment:

  • Test antibody against recombinant ROCK1 and ROCK2 to evaluate isoform specificity

  • For multi-species studies, validate reactivity with ROCK2 orthologs from each target species

  • Available ROCK2 antibodies show validated reactivity across human, mouse, and rat models

• Positive control tissues/cells:

  • Use tissues or cell lines with documented ROCK2 expression levels

  • Compare staining patterns with published literature

Each validation step should be documented with appropriate controls and included in experimental methods sections.

What are the optimal conditions for using ROCK2 antibodies in Western blotting applications?

Optimized Western Blotting Protocol for ROCK2 Detection:

Sample Preparation:

• Extract proteins using RIPA buffer supplemented with phosphatase inhibitors (especially for phospho-ROCK2 detection)

• Load 20-40 μg of total protein per lane (cell lysates) or 50-75 μg (tissue homogenates)

• Include reducing agent (β-mercaptoethanol) in sample buffer due to ROCK2's size (160.9 kDa)

Gel Electrophoresis and Transfer:

• Use 6-8% polyacrylamide gels to resolve the large ROCK2 protein

• Extend transfer time (overnight at 30V in cold room) due to ROCK2's high molecular weight

• Use PVDF membrane (0.45 μm pore size) rather than nitrocellulose for better protein retention

Antibody Incubation:

• Block with 5% non-fat milk or BSA in TBST buffer (use BSA for phospho-specific antibodies)

• Primary antibody dilutions:

  • Monoclonal antibodies (e.g., D-11): 1:500-1:1000

  • Polyclonal antibodies: 1:1000-1:4000 range

• Incubate overnight at 4°C with gentle rocking

• Use HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution

Detection and Troubleshooting:

• For phospho-ROCK2 detection (such as pY256), always include unphosphorylated controls

• Expected band at approximately 160-161 kDa

• If detecting multiple bands, validate specificity using control lysates

• For weak signals, extend exposure time or consider enhanced chemiluminescence substrates

This protocol is based on published methodologies used in ROCK2 research applications and can be further optimized based on specific sample types and antibody characteristics.

How should researchers optimize immunofluorescence protocols for ROCK2 antibodies in different cell types?

Immunofluorescence Optimization Strategy for ROCK2 Detection:

Cell Type-Specific Fixation Methods:

• Epithelial cells: 4% paraformaldehyde (PFA), 10 minutes at room temperature

• Neuronal cells: 2% PFA with 0.1% glutaraldehyde to preserve cytoskeletal structures

• Fibroblasts: Methanol fixation (-20°C, 10 minutes) for better epitope accessibility

Permeabilization Options:

• Standard: 0.1-0.3% Triton X-100 in PBS, 5-10 minutes

• Gentle: 0.05% saponin for preserving membrane structures when studying ROCK2's role in membrane dynamics

• Phospho-epitopes: Use 0.5% NP-40 for improved nuclear penetration when studying phosphorylated ROCK2

Antibody Optimization Table:

Co-localization Studies:

• For stress fiber association: Co-stain with phalloidin (F-actin marker)

• For focal adhesions: Co-stain with anti-paxillin or anti-vinculin

• For Rho GTPase interaction studies: Use anti-RhoA antibodies

• Use confocal microscopy with Z-stack acquisition for spatial relationship analysis

This methodology supports precise localization of ROCK2 in different cellular compartments while maintaining specificity and signal intensity across diverse cell types.

What protocols yield optimal results for ROCK2 immunohistochemistry in formalin-fixed paraffin-embedded tissues?

Optimized IHC Protocol for ROCK2 in FFPE Specimens:

Antigen Retrieval Methods (by tissue type):

• Brain tissue: Citrate buffer (pH 6.0), 95°C for 20 minutes in pressure cooker

• Tumor samples: EDTA buffer (pH 9.0), 95°C for 30 minutes

• Muscle tissue: Tris-EDTA (pH 9.0) with 0.05% Tween-20, 98°C for 20 minutes

Blocking and Antibody Application:

• Block endogenous peroxidase: 3% H₂O₂, 10 minutes

• Protein block: 5-10% normal serum in PBS, 30 minutes

• Primary antibody dilutions:

  • Anti-ROCK2 monoclonal: 1:100-1:200 in antibody diluent

  • Anti-ROCK2 polyclonal: 1:200-1:500 in antibody diluent

• Incubation: Overnight at 4°C in humidified chamber

• Secondary antibody: Species-appropriate HRP-polymer system, 30 minutes at room temperature

Signal Development and Counterstaining:

• DAB chromogen: 5-10 minutes with monitoring

• Counterstain: Mayer's hematoxylin, 1-2 minutes

• Dehydration: Graded ethanol series followed by xylene

• Mounting: Use permanent mounting medium

Critical Quality Controls:

• Positive control: Include bladder carcinoma or renal cell carcinoma sections (known to express ROCK2)

• Negative control: Omit primary antibody on duplicate section

• Absorption control: Pre-incubate antibody with recombinant ROCK2 as specificity control

This protocol has been validated for detection of ROCK2 in paraneoplastic encephalitis and urogenital cancer tissues and can be applied to various tissue types with appropriate optimization of antigen retrieval conditions.

How can researchers effectively use phospho-specific ROCK2 antibodies to study kinase activation states?

Phospho-specific ROCK2 antibodies detect distinct phosphorylation sites that correlate with different activation states of the kinase. These antibodies require specialized handling for optimal results:

Key Phosphorylation Sites and Their Significance:

• Phospho-Ser1366: Associated with ROCK2 activation following RhoA binding; critical for monitoring canonical pathway activation

• Phospho-Y256: Reflects tyrosine kinase-mediated regulation of ROCK2; important in cancer signaling networks

Experimental Design for Phospho-ROCK2 Analysis:

• Sample Preparation Protocol:

  • Rapidly harvest cells/tissues in ice-cold PBS

  • Include phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄, 10 mM β-glycerophosphate)

  • Lyse cells directly in 2X SDS sample buffer for immediate denaturation

  • Heat samples at 95°C for 5 minutes and proceed immediately to electrophoresis

• Activation State Analysis:

  • Compare phospho-ROCK2 to total ROCK2 levels by sequential probing or parallel blots

  • Calculate phospho:total ROCK2 ratio for quantitative assessment

  • Include positive controls: serum-stimulated cells (15% FBS, 30 min) or LPA-treated cells (10 μM, 15 min)

• Inhibitor Studies Design:

  • Pre-treat cells with ROCK inhibitors (Y-27632 or Fasudil) as negative controls

  • Compare selective ROCK2 inhibitors (SR3677) versus pan-ROCK inhibitors

  • Monitor downstream substrates (MYPT1-pT853, MLC-pS19) to confirm functional significance

• Troubleshooting Low Signal Issues:

  • Ensure rapid sample processing (<30 seconds from cell/tissue harvest to lysis)

  • Use fresh phosphatase inhibitors in all buffers

  • For weak signals, consider membrane-based signal enhancement systems

  • For high background, increase blocking time and wash duration

This methodological approach allows researchers to accurately monitor ROCK2 activation status in response to various stimuli, providing insights into signaling pathway dynamics in both normal and pathological states.

What are the most effective strategies for co-immunoprecipitation of ROCK2 and its binding partners?

Optimized Co-Immunoprecipitation Protocol for ROCK2 Complexes:

Lysis Buffer Composition:

• Base buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA

• Detergents: 0.5% NP-40 or 1% CHAPS (preferred for membrane-associated complexes)

• Protease inhibitors: Complete protease inhibitor cocktail

• Phosphatase inhibitors: 10 mM NaF, 1 mM Na₃VO₄, 10 mM β-glycerophosphate

• Stabilizing agents: 10% glycerol, 1 mM DTT

IP Procedure for Different ROCK2 Interaction Partners:

Critical Controls for ROCK2 Co-IP Experiments:

• Input control: 5-10% of lysate used for IP

• Isotype control: Non-specific IgG of same species as ROCK2 antibody

• Antibody-only control: IP procedure without cell lysate

• Competing peptide control: Pre-incubate antibody with immunizing peptide

• Reverse IP: Use antibody against interaction partner to pull down ROCK2

Validation of Interactions:

• Confirm interaction by reciprocal IP (IP with partner, blot for ROCK2)

• Verify physiological relevance by testing interaction after stimulus (e.g., LPA treatment)

• For novel interactions, confirm with alternative methods (proximity ligation assay or FRET)

This detailed protocol, adapted from successful ROCK2 interaction studies, provides a methodological framework for investigating ROCK2 protein complexes while minimizing non-specific binding and maintaining native protein interactions.

What approaches can be used to differentiate between ROCK1 and ROCK2 in experimental systems?

Distinguishing between the highly homologous ROCK1 and ROCK2 isoforms requires careful methodological considerations:

Antibody-Based Differentiation Strategies:

• Epitope selection: Use antibodies targeting the unique C-terminal regions (amino acids 1346-1388 for ROCK2)

• Validation protocol: Test each antibody against recombinant ROCK1 and ROCK2 proteins in parallel Western blots

• Cross-reactivity assessment: Perform peptide competition assays with specific blocking peptides for each isoform

Genetic Approaches for Isoform-Specific Studies:

• siRNA/shRNA design guidelines:

  • Target 3' UTR regions unique to each isoform

  • Validate knockdown efficiency by qRT-PCR with isoform-specific primers

  • Confirm protein reduction by Western blot with validated isoform-specific antibodies

• CRISPR-Cas9 knockout strategy:

  • Design guide RNAs targeting exons unique to ROCK2 (exons 32-36)

  • Screen clones by genomic PCR and confirm by sequencing

  • Validate at protein level using isoform-specific antibodies

Functional Differentiation Assays:

Experimental Controls:

• Use tissues with known differential expression (brain for ROCK2, spleen for ROCK1)

• Include isoform-specific positive controls in all experiments

• For critical experiments, confirm findings using both genetic and pharmacological approaches

This comprehensive approach enables researchers to definitively distinguish between ROCK isoforms, essential for accurately determining their specific roles in physiological and pathological processes.

How can ROCK2 antibodies be used to investigate its role in cancer progression and metastasis?

Methodological Approaches for ROCK2 in Cancer Research:

Tissue Microarray Analysis Protocol:

• Prepare multi-tumor and matched normal tissue arrays

• Perform IHC with validated anti-ROCK2 antibodies

• Score ROCK2 expression using H-score method (intensity × percentage positive cells)

• Correlate with clinicopathological parameters and survival data

Cancer Cell Line Invasion Models:

• Transwell invasion assay with ROCK2 knockdown/overexpression

• 3D spheroid invasion into collagen matrices with live imaging

• Measure effects of ROCK2 inhibition on invadopodia formation

• Quantify changes in cell morphology using immunofluorescence with anti-ROCK2 antibodies

ROCK2 Expression Patterns in Major Cancer Types:

Biomarker Potential Assessment:

• Use phospho-specific antibodies (pSer1366, pY256) to assess activation state

• Develop tissue microarray studies with outcome correlation

• Compare ROCK2 levels in circulating tumor cells versus primary tumors

• Evaluate ROCK2 expression before and after therapeutic intervention

This methodological framework provides a comprehensive approach for investigating ROCK2's role in cancer, from basic expression analysis to functional studies of its contribution to invasive phenotypes.

What protocols are recommended for studying ROCK2 in neurodegenerative and autoimmune disorders?

ROCK2 Analysis in Neurological and Autoimmune Contexts:

Brain Tissue Processing for ROCK2 Immunohistochemistry:

• Fix brain tissue in 4% paraformaldehyde (24h at 4°C)

• Process and embed in paraffin; cut 5 μm sections

• Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Block with 10% normal serum and 0.3% Triton X-100

• Incubate with anti-ROCK2 antibody (1:200 dilution) overnight at 4°C

• Develop using DAB and counterstain with hematoxylin

Co-localization Studies in Neuroinflammatory Conditions:

• Double immunofluorescence protocol for ROCK2 with cell-type markers:

  • Neurons: NeuN or MAP2

  • Astrocytes: GFAP

  • Microglia: Iba1

  • T cells: CD8, CD4

• Use confocal microscopy with z-stack acquisition

• Analyze co-localization using Pearson's correlation coefficient

ROCK2 in Paraneoplastic Encephalitis Assessment:

Methodological Notes for Autoimmune Studies:

• For autoantibody screening, use recombinant ROCK2-transfected HEK293 cells

• Include competitive inhibition experiments with recombinant antigens

• For patient serum studies, dilute 1:100-1:1000 in antibody diluent

• Always include control sera from healthy subjects and disease controls

This comprehensive approach enables detailed investigation of ROCK2's role in neurological and autoimmune disorders, with particular relevance to paraneoplastic syndromes associated with urogenital cancers .

How can researchers optimize ROCK2 antibody-based assays for high-throughput drug screening applications?

ROCK2 Antibody-Based High-Throughput Screening Methodology:

ELISA-Based Inhibitor Screening Protocol:

• Coat 384-well plates with recombinant ROCK2 substrate (MYPT1 fragment)

• Add test compounds with recombinant active ROCK2 enzyme

• Detect substrate phosphorylation using phospho-specific antibodies

• Calculate inhibition relative to positive (Y-27632) and negative controls

Cell-Based Phosphorylation Assays:

• Culture cells in 96-well plates with clear bottoms

• Treat with test compounds (2-4 hours)

• Fix and permeabilize cells in situ

• Probe with phospho-ROCK2 antibodies (Ser1366 or Y256)

• Counterstain nuclei with DAPI

• Quantify using automated fluorescence microscopy

High-Content Imaging Protocol Optimization:

Data Analysis Framework:

• Automated cell segmentation based on nuclear and cytoplasmic markers

• Quantify ROCK2 or phospho-ROCK2 intensity in defined compartments

• Calculate Z-factor for assay quality control (aim for Z' > 0.5)

• Implement machine learning algorithms for phenotypic classification

• Validate hits with orthogonal assays (kinase activity, stress fiber formation)

Troubleshooting Guide for Common HTS Issues:

• High background: Increase blocking time, optimize antibody dilutions

• Poor reproducibility: Standardize cell seeding and treatment times

• Low signal window: Try signal amplification systems or alternative antibody clones

• Edge effects: Use only interior wells or implement humidity control measures

This methodology enables efficient screening of large compound libraries for ROCK2 modulation while maintaining the specificity and quantitative reliability needed for drug discovery applications.

How can researchers implement advanced imaging techniques with ROCK2 antibodies to study its dynamic regulation?

Advanced Imaging Methodologies for ROCK2 Dynamics:

Live-Cell Imaging with Fluorescently Tagged Antibody Fragments:

• Generate Fab fragments from validated anti-ROCK2 antibodies

• Conjugate with cell-permeable fluorophores (e.g., SiR dyes)

• Optimize loading concentration (typically 0.5-2 μM) and incubation time

• Perform time-lapse imaging to visualize ROCK2 redistribution during cellular processes

Super-Resolution Microscopy Protocol:

• Sample preparation: Fix cells with 3% PFA + 0.1% glutaraldehyde

• Primary antibody: Anti-ROCK2 (D-11) at 1:100 dilution

• Secondary antibody: Alexa Fluor 647-conjugated anti-mouse IgG

• Imaging buffer: Oxygen scavenging system with MEA

• Acquisition parameters: 10,000-15,000 frames, 30ms exposure

• Reconstruction: ThunderSTORM or similar algorithm

FRET-Based ROCK2 Activity Sensors:

• Design: CFP-ROCK2 substrate sequence-YFP constructs

• Controls: Include constitutively active and inactive mutants

• Imaging protocol: Excite at 430nm, measure emission at 475nm and 530nm

• Analysis: Calculate emission ratio (530nm/475nm) as ROCK2 activity index

• Validation: Confirm sensor response with ROCK2 inhibitors

Correlative Light-Electron Microscopy (CLEM) Approach:

Technical Recommendations:

• For actin dynamics co-visualization, combine with SiR-Actin or LifeAct probes

• For RhoA-ROCK2 interaction, implement optogenetic RhoA activation during imaging

• Use FRAP (Fluorescence Recovery After Photobleaching) to measure ROCK2 mobility

• Implement adaptive optics for deep tissue imaging in organoids or tissue slices

These advanced imaging approaches enable researchers to visualize ROCK2 dynamics with unprecedented spatiotemporal resolution, providing insights into its regulation and function in various cellular contexts.

What are the recommended approaches for developing quantitative assays to measure ROCK2 activity in complex biological samples?

Quantitative ROCK2 Activity Assay Development:

In Vitro Kinase Assay Protocol:

• Immunoprecipitate ROCK2 using validated antibodies (e.g., D-11)

• Perform kinase reaction with recombinant substrate (MYPT1-fragment)

• Quantify phosphorylation by Western blot or ELISA using phospho-specific antibodies

• Calculate activity relative to standard curve of recombinant active ROCK2

Cell-Based ROCK2 Activity Reporter System:

• Transfect cells with ROCK2 substrate-based FRET biosensor

• Measure baseline FRET ratio in live cells

• Apply experimental treatments

• Quantify changes in FRET ratio as indicator of ROCK2 activity

• Validate with pharmacological inhibitors (Y-27632, SR3677)

Multiplex Phospho-Substrate Analysis:

Mass Spectrometry-Based ROCK2 Activity Profiling:

• Immunoprecipitate ROCK2 from biological samples using specific antibodies

• Perform in vitro kinase reaction with heavy-isotope labeled ATP

• Digest samples and enrich for phosphopeptides

• Analyze by LC-MS/MS to identify and quantify ROCK2 substrates

• Compare phosphorylation patterns across experimental conditions

Assay Validation Strategy:

• Establish Z' factor and intra/inter-assay variability

• Determine linear dynamic range for each biological sample type

• Include specificity controls (ROCK2 inhibitors, ROCK2 knockdown)

• Develop standard operating procedures for sample collection and handling

• Create internal control standards for cross-experiment normalization

This multifaceted approach to ROCK2 activity assessment provides researchers with reliable quantitative tools applicable across various experimental systems, from purified proteins to complex tissues.

What are the key considerations for selecting the most appropriate ROCK2 antibodies for specific research applications?

Comprehensive ROCK2 Antibody Selection Framework:

Application-Specific Selection Criteria:

Technical Specification Assessment:

• Epitope location: N-terminal, internal, or C-terminal region of ROCK2

• Species reactivity: Human, mouse, rat, or multi-species verification

• Clonality: Monoclonal for consistency, polyclonal for sensitivity

• Validated applications: Match to intended experimental use

• Publication record: Evidence of successful use in peer-reviewed literature

Phospho-Specific Antibody Considerations:

• Confirm specific phosphorylation site recognition (Ser1366, Y256)

• Verify phosphorylation state specificity with phosphatase treatments

• Consider antibody pairs for total/phospho detection in the same experiment

• Evaluate cross-reactivity with related kinases (especially ROCK1)

Special Research Context Recommendations:

• For isoform discrimination: Choose antibodies targeting unique C-terminal regions

• For neurodegenerative research: Select antibodies validated in brain tissue

• For cancer studies: Use antibodies with demonstrated specificity in relevant tumor types

• For autoimmunity research: Consider antibodies that don't compete with patient autoantibodies

This systematic approach to ROCK2 antibody selection ensures optimal results across diverse experimental applications while minimizing technical artifacts and maximizing research reproducibility.

What are the emerging trends and future directions in ROCK2 antibody research and applications?

Future Directions in ROCK2 Antibody Technology and Applications:

Emerging Antibody Technologies:

• Single-domain nanobodies: Developing camelid-derived ROCK2 nanobodies for improved tissue penetration and intracellular delivery

• Bispecific antibodies: Creating antibodies that simultaneously target ROCK2 and its binding partners for studying protein-protein interactions

• Conditionally stable antibody fragments: Engineering antibody fragments that report on ROCK2 conformational changes upon activation

• Intrabodies: Developing intracellularly expressed antibody fragments for real-time visualization of endogenous ROCK2

Novel Application Areas:

Integration with Advanced Technologies:

• Combining with CRISPR-mediated endogenous tagging for validated antibody targets

• Implementing machine learning algorithms for automated analysis of ROCK2 distribution patterns

• Developing antibody-based biosensors for continuous ROCK2 activity monitoring

• Creating antibody-guided proximity labeling for ROCK2 interaction network mapping

Translational Research Directions:

• Developing standardized ROCK2 immunoassays for clinical samples

• Creating companion diagnostic tests for ROCK2-targeting therapeutics

• Establishing ROCK2 antibody panels for cancer subtype classification

• Exploring ROCK2 autoantibodies as biomarkers in paraneoplastic and autoimmune conditions

These forward-looking approaches represent the cutting edge of ROCK2 antibody research, promising to expand our understanding of ROCK2 biology and its implications in disease while opening new avenues for diagnostic and therapeutic development.