ROCK2 Antibody, HRP conjugated

What is ROCK2 and what cellular functions does it regulate?

ROCK2 (Rho-associated coiled-coil containing protein kinase 2) is a serine/threonine kinase that functions as a key regulator of actin cytoskeleton and cell polarity. It plays critical roles in multiple cellular processes including:

• Regulation of smooth muscle contraction

• Organization of actin cytoskeleton

• Formation of stress fibers and focal adhesions

• Neurite retraction

• Cell adhesion and motility

• Phosphorylation of various substrates including ADD1, BRCA2, CNN1, EZR, DPYSL2, EP300, MSN, MYL9/MLC2, NPM1, RDX, PPP1R12A and VIM

In the central nervous system, ROCK2 is extensively expressed in the brain and spinal cord, with expression levels increasing with age. ROCK2 inhibition has been shown to attenuate axonal degeneration, prevent apoptosis, and stimulate neurite outgrowth . At the molecular level, inhibiting ROCK2 reduces caspase-3 and calpain activity, enhances autophagic flux, and reduces acute axonal degeneration .

What is the molecular weight and cellular localization of ROCK2?

Regarding cellular localization, ROCK2 is primarily found in the cytoplasm and also associates with the cell membrane. It specifically interacts with actin microfilaments and the plasma membrane . This localization is crucial for its role in regulating cytoskeletal dynamics and cell morphology.

What species reactivity can I expect from commercially available ROCK2 antibodies?

Most commercially available ROCK2 antibodies demonstrate cross-reactivity across multiple mammalian species. Based on the available data, ROCK2 antibodies typically react with:

This cross-reactivity is advantageous as it allows researchers to use the same antibody across different model organisms, facilitating comparative studies between species.

What are the optimal protocols for using ROCK2 antibodies in Western blot applications?

For optimal Western blot results with ROCK2 antibodies, the following protocol considerations are recommended:

• Sample preparation: Prepare lysates from appropriate cell lines known to express ROCK2 (e.g., HepG2, COLO 205, DA3, CH-1, PC-12) .

• Gel electrophoresis: Use reducing conditions when separating proteins.

• Transfer conditions: Transfer proteins to PVDF membrane for optimal results.

• Blocking: Block with 5% non-fat dried milk in TBST for 1 hour at room temperature .

• Primary antibody incubation:

  • For AF4790: Use at 1 μg/mL

  • For ab125025: Use at 1:10000 dilution

  • For E-AB-52117: Use at 1:500-1:2000 dilution

• Secondary antibody:

  • For rabbit primary antibodies: Use HRP-conjugated anti-rabbit IgG at 1:20000 dilution

  • For goat primary antibodies: Use HRP-conjugated anti-goat IgG secondary antibody

• Detection: Use an appropriate chemiluminescent substrate compatible with HRP-conjugated antibodies.

• Expected result: A specific band should be detected at approximately 160-161 kDa .

How should I optimize ROCK2 antibody dilutions for different experimental applications?

Optimal antibody dilution varies based on the specific application, antibody clone, and detection method. The following table provides recommended dilutions based on available data:

*Specific dilution not provided in the search results

When using HRP-conjugated secondary antibodies for detection, recommended dilutions typically range from 1:2000 to 1:20000, with higher dilutions (1:20000) often used with highly sensitive detection systems .

For precise optimization, a titration experiment is recommended where you test a range of dilutions to determine the concentration that provides the best signal-to-noise ratio for your specific experimental conditions.

What controls should I include when using ROCK2 antibodies in my experiments?

To ensure experimental rigor when working with ROCK2 antibodies, include the following controls:

• Positive controls: Cell lines known to express ROCK2 such as:

  • HepG2 (human hepatocellular carcinoma)

  • COLO 205 (human colorectal adenocarcinoma)

  • DA3 (mouse myeloma)

  • CH-1 (mouse B cell lymphoma)

  • PC-12 (rat adrenal pheochromocytoma)

  • L6 cells

  • RAW264.7 cells

• Negative controls:

  • ROCK2 knockout cell lysates (e.g., Human ROCK2 knockout HeLa cell lysate ab257643)

  • Samples treated with ROCK2 siRNA (validated to reduce ROCK2 expression)

  • Secondary antibody only (no primary antibody)

• Loading controls: Include antibodies against housekeeping proteins such as GAPDH to normalize protein loading .

• Specificity controls:

  • For antibodies that recognize multiple species, test cross-reactivity with recombinant proteins if available

  • Include competitive blocking with the immunogen peptide if available

Incorporating these controls helps validate specificity and ensures that observed signals are genuinely attributable to ROCK2 rather than non-specific binding or technical artifacts.

How can I study ROCK2 interactions with other proteins in signaling pathways?

To investigate ROCK2 protein interactions, several methodological approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate using anti-ROCK2 antibodies followed by Western blot for suspected interacting partners

  • Alternatively, immunoprecipitate with antibodies against suspected binding partners followed by ROCK2 detection

  • This approach was successfully used to demonstrate ROCK2 interaction with p22phox in THP1 cells

• Dot-blot assays for direct interaction studies:

  • Apply recombinant ROCK2 protein fragments to a nitrocellulose membrane

  • Probe with recombinant versions of suspected binding partners

  • Detect interactions using appropriate antibodies and HRP-conjugated secondary antibodies

  • This approach revealed that p22phox specifically interacts with ROCK2 (400-967) fragment but not with His-ROCK2 (11-552) or ROCK2 (968-1,388)

• siRNA-mediated knockdown:

  • Transfect cells with ROCK2-specific siRNA to reduce its expression

  • Assess effects on binding partners and downstream signaling

  • Validate specificity by comparing with control siRNA or ROCK1 siRNA

• Inhibitor studies:

  • Use ROCK2-selective inhibitors (e.g., KD025) versus pan-ROCK inhibitors (e.g., Y27632)

  • Compare differential effects on protein interactions and signaling pathways

  • This approach helps distinguish ROCK2-specific functions from those shared with ROCK1

When using HRP-conjugated detection systems in these experiments, carefully optimize antibody dilutions and incubation times to minimize background while maintaining sensitivity.

What roles does ROCK2 play in NADPH oxidase activation and ROS production?

Research has revealed a previously unrecognized role for ROCK2 in regulating NADPH oxidase activation and reactive oxygen species (ROS) production in monocytes:

• Direct protein interactions:

  • ROCK2 directly interacts with p22phox, a component of the NADPH oxidase complex

  • This interaction was identified through mass spectrometry analysis of p22phox immunoprecipitates

  • The interaction specifically involves the ROCK2 (400-967) fragment

• Phosphorylation of NADPH oxidase components:

  • ROCK2 phosphorylates p47phox, a cytosolic component of the NADPH oxidase

  • This phosphorylation is crucial for NADPH oxidase assembly and activation

• Functional significance in ROS production:

  • ROCK2 inhibitors reduce ROS production in human monocytes

  • siRNA-mediated knockdown of ROCK2 in THP1 cells significantly decreases ROS production in response to various stimuli (fMLF, PMA, and Op. Zymosan)

  • This effect is specific to ROCK2, as ROCK1 siRNA did not inhibit ROS production

• Methodological considerations for studying this pathway:

  • Use fluorescent or chemiluminescent probes to measure ROS production

  • Include both ROCK2-selective inhibitors and pan-ROCK inhibitors to distinguish isoform-specific effects

  • Validate with genetic approaches (siRNA, CRISPR) to confirm specificity

This research highlights the potential of ROCK2 as a therapeutic target in conditions involving dysregulated ROS production and inflammatory responses.

How can I distinguish between ROCK1 and ROCK2 in experimental settings?

Distinguishing between the highly homologous ROCK1 and ROCK2 isoforms requires careful experimental design:

• Antibody selection:

  • Use antibodies that specifically recognize unique epitopes in each isoform

  • Validate antibody specificity using knockout or knockdown models

  • For example, ab125025 has been validated using ROCK2 knockout HeLa cells

• Selective inhibitors:

  • Y27632 inhibits both ROCK1 and ROCK2 (non-selective)

  • KD025 is more selective for ROCK2

  • Compare effects of selective versus non-selective inhibitors to distinguish isoform-specific functions

• siRNA/shRNA approaches:

  • Use siRNA targeting specific sequences unique to either ROCK1 or ROCK2

  • Include both ROCK1 and ROCK2 siRNAs in parallel experiments to compare effects

  • This approach demonstrated that ROCK2, but not ROCK1, siRNA inhibited ROS production in THP1 cells

• Expression patterns:

  • ROCK2 is extensively expressed in brain and spinal cord, with expression increasing with age

  • Consider tissue-specific expression differences when designing experiments

• Functional readouts:

  • Some functions may be specifically attributed to one isoform

  • For example, ROCK2 specifically interacts with p22phox and regulates NADPH oxidase in monocytes

When using HRP-conjugated detection systems, optimize conditions for each primary antibody separately, as the optimal dilution and incubation times may differ between ROCK1 and ROCK2 antibodies.

Why might I observe unexpected band patterns when using ROCK2 antibodies in Western blot?

Several factors can contribute to unexpected band patterns when detecting ROCK2:

• Post-translational modifications:

  • ROCK2 undergoes phosphorylation and potentially other modifications

  • These modifications can alter protein mobility on SDS-PAGE

  • Consider using phosphatase treatment of samples if you suspect phosphorylation is causing band shifts

• Protein degradation:

  • Include protease inhibitors in lysis buffers

  • Avoid repeated freeze-thaw cycles of samples

  • Keep samples cold during preparation

• Alternative splicing:

  • ROCK2 may have splice variants in certain tissues or conditions

  • Consult transcript databases to identify potential variants

• Cross-reactivity:

  • Some antibodies may cross-react with related proteins

  • Validate specificity using knockout or knockdown controls

• Sample processing effects:

  • Different sample buffer compositions can affect protein migration

  • Ensure consistent reducing conditions across experiments

As noted in the Elabscience antibody datasheet: "The actual band is not consistent with the expectation. Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

How can I optimize signal-to-noise ratio when using HRP-conjugated secondary antibodies with ROCK2 primary antibodies?

To achieve optimal signal-to-noise ratio when using HRP-conjugated detection systems:

• Antibody dilutions:

  • Primary antibody: Start with manufacturer's recommended dilution (e.g., 1:10000 for ab125025)

  • HRP-conjugated secondary antibody: Typically 1:2000 to 1:20000 based on sensitivity requirements

• Blocking optimization:

  • Use 5% non-fat dried milk in TBST for 1 hour at room temperature

  • For phospho-specific detection, consider 5% BSA instead of milk

  • Extend blocking time if background remains high

• Wash protocols:

  • Increase wash duration and number of washes (e.g., 5 x 5 minutes with TBST)

  • Ensure gentle but thorough agitation during washes

• Substrate selection:

  • Match substrate sensitivity to expression level of target

  • For low expression, use high-sensitivity ECL substrates

  • For abundant proteins, standard ECL is sufficient

  • Consider exposure time optimization

• Buffer composition:

  • Use optimized immunoblot buffer groups as recommended (e.g., Immunoblot Buffer Group 1 for AF4790)

• Antibody incubation conditions:

  • Primary antibody: Overnight at 4°C or 1-2 hours at room temperature

  • Secondary antibody: Typically 1 hour at room temperature

Careful optimization of these parameters will help achieve the clearest specific signal while minimizing background.

What methodological approaches can I use to study ROCK2 inhibition effects on cellular processes?

To investigate the functional consequences of ROCK2 inhibition, several complementary approaches can be employed:

• Pharmacological inhibition:

  • Use selective ROCK2 inhibitors (e.g., KD025) versus pan-ROCK inhibitors (Y27632)

  • Establish dose-response relationships and treatment durations

  • Monitor effects on:

    • Cytoskeletal organization

    • Cell migration and adhesion

    • ROS production

    • Phosphorylation of downstream targets

• Genetic knockdown/knockout:

  • siRNA-mediated knockdown of ROCK2 (validated to reduce protein expression)

  • CRISPR/Cas9-mediated knockout for complete elimination

  • Validate specificity by comparing with ROCK1 targeting

• Functional readouts:

  • For ROS production: Use fluorescent probes or chemiluminescence assays

  • For cytoskeletal changes: Immunofluorescence staining of F-actin and focal adhesions

  • For signaling pathway activation: Phospho-specific antibodies against ROCK2 substrates

  • For axonal degeneration: Neurite outgrowth assays

• Molecular mechanism studies:

  • Monitor effects on interactions between ROCK2 and binding partners (e.g., p22phox)

  • Assess phosphorylation status of ROCK2 substrates (e.g., p47phox)

  • Examine effects on enzyme activities (e.g., caspase-3, calpain, autophagic flux)

Research has shown that ROCK2 inhibition produces significant biological effects including attenuation of axonal degeneration, prevention of apoptosis, and stimulation of neurite outgrowth . Additionally, ROCK2 inhibition reduces ROS production in monocytes by affecting NADPH oxidase activation .

How can I validate the specificity of my ROCK2 antibody?

Rigorous validation of ROCK2 antibody specificity is essential for reliable experimental results:

• Knockout/knockdown controls:

  • Use ROCK2 knockout cell lysates (e.g., Human ROCK2 knockout HeLa cell line ab265679)

  • Compare with wild-type cells to confirm specific band disappearance

  • Use siRNA to reduce ROCK2 expression and confirm decreased signal intensity

• Immunodepletion:

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

  • This should eliminate specific signal if the antibody is truly specific

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of ROCK2

  • Consistent results with different antibodies increase confidence in specificity

• Cross-species reactivity:

  • Test antibody across multiple species (human, mouse, rat)

  • Consistent detection at the predicted molecular weight supports specificity

• Mass spectrometry validation:

  • Immunoprecipitate ROCK2 and confirm identity by mass spectrometry

  • This approach was successfully used to identify ROCK2 as a p22phox-interacting protein

When using HRP-conjugated detection systems, include appropriate secondary antibody-only controls to assess non-specific binding of the secondary antibody.

What are the considerations for using ROCK2 antibodies in co-localization studies with other proteins?

When designing co-localization studies to investigate ROCK2 interactions with other proteins:

• Antibody compatibility:

  • Select primary antibodies from different host species to allow simultaneous detection

  • For example, rabbit anti-ROCK2 can be paired with mouse antibodies against potential binding partners

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider brightness and photostability for optimal imaging

• Sample preparation:

  • Optimize fixation methods (paraformaldehyde is commonly used)

  • Test different permeabilization reagents (Triton X-100, saponin, methanol)

  • Consider antigen retrieval if necessary

• Controls for co-localization:

  • Include single-stained samples for each antibody to establish specificity

  • Use known interacting proteins as positive controls

  • Include non-interacting proteins as negative controls

• Image acquisition and analysis:

  • Use confocal microscopy for optimal spatial resolution

  • Collect Z-stacks to examine co-localization in three dimensions

  • Apply quantitative co-localization analysis (Pearson's coefficient, Manders' coefficient)

• Validation with functional assays:

  • Complement imaging with biochemical interaction assays (co-IP, proximity ligation)

  • Test if disrupting the interaction affects localization patterns

ROCK2 co-localization with p22phox in intact monocytes has been demonstrated and provides insight into their functional interaction in regulating NADPH oxidase activity .

By carefully addressing these considerations, researchers can generate robust co-localization data that provides meaningful insights into ROCK2's interactions and functions within cells.