ROCK1 Antibody

What is ROCK1 and what are its key biological functions?

ROCK1 (Rho-associated, coiled-coil containing protein kinase 1), also known as p160ROCK and NY-REN-35, belongs to the protein kinase superfamily and the AGC Ser/Thr protein kinase family . With a calculated molecular weight of 158 kDa (1354 amino acids), ROCK1 typically appears as a 150-160 kDa band on Western blots .

ROCK1 functions as:

• A key modulator of cytoskeletal actin organization and cell polarity

• A crucial regulator of cell adhesion and motility mechanisms

• A physiological regulator of PTEN, where it directly binds PTEN in response to receptor activation and is essential for PTEN phosphorylation and stability

• A suppressor of inflammatory cell migration for macrophages and neutrophils during acute inflammation

• A component in the RhoA/ROCK1 signaling pathway that determines cell fate by promoting stress granule formation or initiating apoptosis

Dysregulation of ROCK1 has been linked to cancer metastasis, cardiovascular diseases, and neurological disorders, making it an important subject for therapeutic research .

What types of ROCK1 antibodies are available for research applications?

Based on the search results, researchers can access several types of ROCK1 antibodies:

• By production method:

  • Polyclonal antibodies (e.g., 21850-1-AP) - derived from multiple B cell lineages

  • Monoclonal antibodies (e.g., CAB11158) - produced from a single B cell clone, offering higher specificity and consistency

• By conjugation status:

  • Unconjugated antibodies for standard applications

  • Conjugated antibodies, such as CoraLite® Plus 488 Fluorescent Dye-conjugated (CL488-21850) with excitation/emission maxima wavelengths of 493 nm / 522 nm for fluorescence applications

• By host species:

  • Rabbit-derived anti-ROCK1 antibodies are common among the search results

What applications can ROCK1 antibodies be used for in scientific research?

ROCK1 antibodies can be utilized across multiple experimental applications:

Additionally, ROCK1 antibodies have been used in specialized assays such as ROCK1 activity assays where immunoprecipitated complexes are assessed for their ability to phosphorylate MYPT1 (myosin phosphatase target subunit 1) .

What are the optimal sample types and validated reactivity for ROCK1 antibodies?

When designing experiments with ROCK1 antibodies, researchers should consider validated sample types:

Cell Lines with Confirmed Positive Detection:

• Human: A431, HeLa, HEK-293T, Jurkat, Ramos, HT-29, Hep G2

• Rodent: C6 (rat), NIH/3T3 (mouse)

Tissue Samples with Confirmed Positive Detection:

• Human lymphoma tissue (for IHC applications)

• Mouse lung tissue

Species Reactivity:
Most ROCK1 antibodies in the search results demonstrate cross-reactivity with:

• Human ROCK1

• Mouse ROCK1

• Rat ROCK1

This cross-reactivity makes these antibodies versatile for comparative studies across species, though researchers should validate antibody performance in their specific experimental system .

How should I optimize ROCK1 antibody use for immunohistochemistry applications?

For optimal IHC results with ROCK1 antibodies:

• Antigen Retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Dilution Optimization:

  • Begin with manufacturer-recommended dilution (typically 1:50-1:500)

  • Conduct titration experiments to determine optimal concentration for your specific tissue type

  • Prepare working dilutions fresh before use

• Controls:

  • Positive control: Use human lymphoma tissue, which has been validated for ROCK1 detection

  • Negative control: Include sections processed without primary antibody

  • Consider using ROCK1 knockout/knockdown samples when available

• Detection Systems:

  • Use detection systems appropriate for rabbit IgG primaries

  • For fluorescent detection, select secondary antibodies with minimal spectral overlap if performing multiplex staining

What methodological approaches can be used to study ROCK1 activation rather than just protein expression?

To assess ROCK1 activity rather than mere presence:

• ROCK1 Kinase Activity Assay:

  • Immunoprecipitate ROCK1 from cell lysates using anti-ROCK1 antibody

  • Incubate immunoprecipitated complexes with ROCK1 substrate protein (MYPT1)

  • Detect phosphorylated MYPT1 via Western blotting with anti-phospho-MYPT1 antibody

  • The amount of phosphorylated MYPT1 indicates ROCK1 activity

• Upstream Activation Analysis:

  • Assess RhoA activation (ROCK1's upstream activator) using GST-Rhotekin pulldown assays

  • Active RhoA can be detected by Western blotting the pulldown samples with anti-RhoA antibody

• Phosphorylation Status of ROCK1 Substrates:

  • Monitor phosphorylation of known ROCK1 substrates in cellular contexts

  • Use phospho-specific antibodies against ROCK1 targets to assess kinase activity indirectly

• Inhibitor Studies:

  • Use specific ROCK inhibitors like Y-27632 as experimental controls

  • Compare protein phosphorylation patterns between inhibitor-treated and untreated samples

How can ROCK1 antibodies be used to investigate the interaction between ROCK1 and PTEN in inflammatory responses?

Research has revealed ROCK1 as a physiological regulator of PTEN that suppresses excessive recruitment of inflammatory cells. To investigate this interaction:

• Co-immunoprecipitation (Co-IP):

  • Use anti-ROCK1 antibodies to immunoprecipitate protein complexes

  • Probe immunoprecipitates for PTEN via Western blotting

  • This approach can identify physical interaction between ROCK1 and PTEN in response to receptor activation

• Phosphorylation Analysis:

  • Assess PTEN phosphorylation status in wild-type versus ROCK1-deficient models

  • Use anti-phospho-serine antibodies to detect PTEN phosphorylation

  • Research shows that ROCK1 deficiency results in significantly impaired PTEN phosphorylation, stability, and activity

• Downstream Pathway Analysis:

  • Monitor activation of PTEN downstream targets (PIP3, AKT, GSK-3β, cyclin D1)

  • Compare activation patterns between wild-type and ROCK1-deficient systems

  • ROCK1 deficiency leads to increased activation of these downstream targets

• Migration Assays:

  • Conduct in vitro migration assays using bone marrow-derived macrophages (BMMs) and neutrophils

  • Compare migratory capacity on matrices like fibronectin between wild-type and ROCK1-deficient cells

  • Research shows ROCK1-deficient macrophages demonstrate significantly increased migration

• In Vivo Recruitment Studies:

  • Challenge mice with inflammatory stimuli (e.g., thioglycollate)

  • Compare recruitment of inflammatory cells between wild-type and ROCK1-deficient mice

  • Studies show increased recruitment of ROCK1-deficient macrophages (4 days post-stimulation) and neutrophils (4 hours post-stimulation)

What role does the RhoA/ROCK1 pathway play in stress granule formation, and how can researchers investigate this mechanism?

The RhoA/ROCK1 signaling pathway has been identified as a determinant of cell fate by promoting stress granule (SG) formation or initiating apoptosis. Researchers can investigate this role using:

• RhoA Activation Assay:

  • Use GST-Rhotekin pulldown to isolate active RhoA from control or stress-exposed cells

  • Detect active RhoA via Western blotting with anti-RhoA antibody

  • This identifies changes in RhoA activation status under different stress conditions

• ROCK1 Activity Assessment:

  • Immunoprecipitate ROCK1 from cells using anti-ROCK1 antibodies

  • Incubate immunoprecipitates with MYPT1 substrate

  • Detect phosphorylated MYPT1 via Western blotting with specific anti-phospho-MYPT1 antibody

  • This quantifies ROCK1 activity under different experimental conditions

• Stress Granule Analysis:

  • Use anti-TIA-1 antibodies (a stress granule marker) for co-immunoprecipitation studies

  • Investigate ROCK1 localization in stress granules via immunofluorescence

  • Apply ROCK1 inhibitors (e.g., Y-27632) or RhoA inhibitors (e.g., C3 exotoxin) to assess their impact on stress granule formation

• Comparative Analysis with JNK Pathway:

  • Investigate the relationship between ROCK1 activity and JNK activation

  • Use anti-JNK and anti-phospho-JNK antibodies to assess JNK pathway activation in relation to ROCK1 activity

  • This helps understand the interplay between these stress-responsive pathways

How can ROCK1 antibodies be used to study the role of ROCK1 in cancer progression and metastasis?

ROCK1 is implicated in cancer progression, with altered expression in malignant phenotypes. Researchers can investigate this role using:

• Expression Profiling:

  • Compare ROCK1 mRNA and protein expression between normal and malignant cells

  • Use Western blotting, immunohistochemistry, and qPCR to quantify differences

  • Research has shown increased ROCK1 mRNA in some malignant cells (e.g., T4-2 cells) compared to non-malignant counterparts (e.g., S1 cells)

• Reversion Studies:

  • Treat malignant cells with inhibitors (ROCK inhibitor Y-26732, MEK inhibitor PD98059, or EGFR inhibitor AG1478)

  • Assess changes in ROCK1 expression and cellular phenotype

  • Research shows these inhibitors can decrease ROCK1 mRNA and revert malignant phenotypes

• 3D Culture Systems:

  • Establish 3D culture models that better recapitulate physiological conditions

  • Compare ROCK1 expression and function between 2D and 3D cultures

  • Research indicates observations from 3D cultures have greater clinical relevance than 2D cultures for studying ROCK1 in cancer

• Cellular Function Assays:

  • Assess cell migration, invasion, and cytoskeletal organization

  • Correlate these parameters with ROCK1 expression and activity

  • This helps establish the functional significance of ROCK1 in cancer progression

What are common pitfalls when working with ROCK1 antibodies, and how can they be addressed?

Researchers may encounter several challenges when working with ROCK1 antibodies:

• Specificity Issues:

  • Problem: Cross-reactivity with ROCK2 or other kinases

  • Solution: Use antibodies raised against unique regions of ROCK1; conduct validation with ROCK1 knockout/knockdown samples

• Variable Molecular Weight Detection:

  • Problem: ROCK1 may appear at weights other than the expected 150-160 kDa

  • Solution: Be aware that post-translational modifications, degradation, or splice variants may alter migration patterns; use appropriate molecular weight markers and positive controls

• Storage and Stability Issues:

  • Problem: Loss of antibody activity over time

  • Solution: Store antibodies according to manufacturer recommendations (typically -20°C); aliquot to avoid freeze-thaw cycles; add preservatives like glycerol as specified

• Background in Immunostaining:

  • Problem: High background in IF/ICC or IHC applications

  • Solution: Optimize blocking conditions; increase washing steps; test different dilutions; consider antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

How should researchers validate ROCK1 antibody specificity for their particular experimental system?

Comprehensive validation strategies include:

• Gene Knockdown/Knockout Controls:

  • Use ROCK1 siRNA, shRNA, or CRISPR-Cas9 techniques to reduce/eliminate ROCK1 expression

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

  • A specific antibody should show reduced/absent signal in knockdown/knockout samples

• Multiple Antibody Comparison:

  • Test multiple antibodies targeting different epitopes of ROCK1

  • Consistent results across different antibodies support specific detection

  • Discrepancies may indicate non-specific binding or epitope-specific effects

• Recombinant Protein Controls:

  • Use purified recombinant ROCK1 as a positive control

  • This can establish appropriate molecular weight and signal intensity benchmarks

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific antibody signal should be significantly reduced or eliminated

• Cross-Species Reactivity Assessment:

  • If working with non-human samples, confirm antibody reactivity with the species of interest

  • The search results indicate that many ROCK1 antibodies react with human, mouse, and rat samples, but validation in your specific system is still recommended

What are the optimal storage and handling conditions for maintaining ROCK1 antibody performance?

For maximum antibody stability and performance:

• Storage Temperature:

  • Store at -20°C as recommended by manufacturers

  • Aliquoting is typically unnecessary for -20°C storage but may be beneficial for frequently used antibodies

• Storage Buffer Components:

  • Common storage buffers include:

    • PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

    • PBS with 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3

  • These components protect antibody structure and prevent microbial growth

• Light Sensitivity:

  • For fluorescent-conjugated antibodies (e.g., CL488-21850), avoid exposure to light

  • Store in amber tubes or wrap containers in aluminum foil

• Thawing and Handling:

  • Thaw antibodies on ice or at 4°C

  • Mix gently by inversion or finger-flicking; avoid vortexing

  • Keep antibodies on ice during experimental procedures

• Shelf Life:

  • Most ROCK1 antibodies are stable for one year after shipment when stored properly

  • Monitor for signs of degradation such as precipitation or loss of activity

What positive and negative controls should be included when studying ROCK1 in different experimental contexts?

Appropriate controls enhance the reliability of ROCK1 research:

For Western Blot Applications:

• Positive controls: Lysates from A431, HeLa, HEK-293T, Jurkat, NIH/3T3, Ramos, or C6 cells, which have validated ROCK1 expression

• Loading controls: Actin, GAPDH, or other housekeeping proteins to normalize protein loading

• Treatment controls: Samples treated with ROCK inhibitors (Y-27632) to demonstrate specificity

For Immunostaining Applications:

• Positive tissue controls: Human lymphoma tissue for IHC

• Procedural negative controls: Samples processed without primary antibody

• Biological negative controls: Tissues/cells with ROCK1 knockdown/knockout

For Functional Studies:

• Pharmacological controls: Compare results with ROCK inhibitors (Y-27632) and RhoA inhibitors (C3 exotoxin)

• Genetic controls: Compare wild-type with ROCK1-deficient models

• Pathway controls: Include experiments assessing upstream (RhoA) and downstream (MYPT1 phosphorylation) components

How should researchers interpret changes in ROCK1 expression versus ROCK1 activity in experimental results?

When analyzing ROCK1 in experimental systems, distinguishing between expression and activity is crucial:

What considerations should researchers keep in mind when comparing 2D versus 3D culture systems for ROCK1 studies?

Research indicates important differences between 2D and 3D systems when studying ROCK1:

• Physiological Relevance:

  • Observations from 3D cultures have greater clinical relevance than those from 2D cultures

  • 2D culture findings may miss or contradict physiological or clinical observations

• Gene Expression Patterns:

  • ROCK1 mRNA expression patterns differ between 2D and 3D culture systems

  • Malignant cells (e.g., T4-2) show increased ROCK1 expression compared to non-malignant cells (e.g., S1)

• Response to Inhibitors:

  • Cells in 3D culture may respond differently to ROCK inhibitors than in 2D

  • Treatment with ROCK inhibitor (Y-26732), MEK inhibitor (PD98059), or EGFR inhibitor (AG1478) can decrease ROCK1 mRNA in 3D models

• Cytoskeletal Organization:

  • ROCK1's effects on actin organization are particularly important in 3D environments

  • Gene expression profiling reveals increased expression of actin organization genes in some malignant cells

• Experimental Design Recommendations:

  • Validate key findings in both 2D and 3D systems when possible

  • Consider 3D culture models for more physiologically relevant assessment of ROCK1 function

  • Use appropriate matrices (e.g., Matrigel, collagen) that mimic in vivo environments