ROCK2 Antibody, Biotin conjugated

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

ROCK2 (Rho-associated protein kinase 2) is a key regulatory protein kinase that plays crucial roles in actin cytoskeleton organization and cell polarity. It functions as a central mediator in several essential cellular processes including smooth muscle contraction, stress fiber formation, focal adhesion formation, neurite retraction, and cellular motility. ROCK2 exerts these effects through phosphorylation of multiple downstream targets including ADD1, BRCA2, CNN1, EZR, DPYSL2, EP300, MSN, MYL9/MLC2, NPM1, RDX, PPP1R12A, and VIM. Additionally, ROCK2 phosphorylates SORL1 and IRF4, further expanding its regulatory network . Recent research has revealed ROCK2's involvement in the negative regulation of angiogenic endothelial cell activation induced by VEGF, as well as its positive regulatory effects on MAPK signaling during myogenic differentiation .

What distinguishes biotin-conjugated ROCK2 antibodies from other formats?

Biotin-conjugated ROCK2 antibodies feature a covalent attachment of biotin molecules to the antibody structure, enabling enhanced detection capabilities through the high-affinity biotin-avidin system. This conjugation offers significant advantages in detection sensitivity and versatility across multiple applications. The biotin tag allows for signal amplification through subsequent binding with avidin/streptavidin conjugated to enzymes (like HRP), fluorochromes, or other detection molecules . Importantly, biotin-conjugated formats are specifically designed for multiplex detection systems, allowing researchers to simultaneously examine multiple targets in complex experimental setups while maintaining high specificity for ROCK2 .

What cellular locations does ROCK2 typically occupy?

ROCK2 predominantly localizes to the cytoplasm and cell membrane, reflecting its central role in cytoskeletal regulation and signal transduction . This subcellular distribution is consistent with its function in modulating actin dynamics, stress fiber formation, and cell contractility. The membrane association is particularly important for ROCK2's interaction with membrane-bound RhoA, which activates ROCK2 and initiates its kinase activity toward downstream substrates involved in cytoskeletal rearrangement .

What are the validated applications for biotin-conjugated ROCK2 antibodies?

Based on current research literature and manufacturer specifications, biotin-conjugated ROCK2 antibodies have been validated for several key applications:

• Western Blotting (WB): Recommended dilution ranges from 1:300 to 1:5000 depending on specific antibody formulation and experimental conditions .

• Enzyme-Linked Immunosorbent Assay (ELISA): Particularly effective in sandwich ELISA formats where the biotin-conjugated antibody serves as the detection antibody in combination with a capture antibody specific to ROCK2 .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Though less commonly documented specifically for biotin-conjugated formats, the underlying antibody clones have demonstrated reactivity in these applications .

The choice of application should be guided by the specific experimental questions and the validated performance characteristics of the particular antibody clone being used .

How should ELISA protocols be optimized when using biotin-conjugated ROCK2 antibodies?

For optimal ELISA performance with biotin-conjugated ROCK2 antibodies, researchers should follow this methodological approach:

• Plate Preparation: Use a microplate pre-coated with a primary antibody specific to human ROCK2 (capture antibody).

• Sample Application: Apply standards or samples to appropriate wells and incubate at 37°C for approximately 80 minutes to allow ROCK2 binding.

• Antibody Incubation: After washing, add the biotin-conjugated ROCK2 antibody working solution (100 μL per well) and incubate at 37°C for 50 minutes.

• Signal Development: Apply streptavidin-HRP working solution (100 μL per well) after washing, incubate at 37°C for 50 minutes, then wash thoroughly (5 times recommended).

• Detection: Add TMB substrate solution (90 μL per well) and incubate at 37°C for 20 minutes in darkness, then add 50 μL of stop solution.

• Analysis: Measure optical density at 450 nm (±10 nm) and calculate ROCK2 concentration by comparison to the standard curve .

This sandwich ELISA configuration provides excellent specificity, as signal generation requires both the capture antibody and the biotin-conjugated detection antibody to bind to ROCK2, greatly reducing background and increasing sensitivity .

What is the optimal storage condition for maintaining biotin-conjugated ROCK2 antibody activity?

To maintain optimal activity of biotin-conjugated ROCK2 antibodies, store at -20°C for up to 12 months in appropriate buffer conditions. The recommended storage buffer typically contains 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol . The glycerol component prevents freeze-thaw damage, while BSA provides stability and reduces non-specific binding. For working solutions, minimize repeated freeze-thaw cycles by preparing single-use aliquots. When handling the antibody, maintain cold chain integrity and avoid contamination by using sterile techniques .

What species reactivity profiles are available for biotin-conjugated ROCK2 antibodies?

Available biotin-conjugated ROCK2 antibodies demonstrate varied species reactivity profiles depending on the specific clone and manufacturer. Based on the provided information, several options exist:

Researchers should select antibodies based on their target species, with consideration for cross-reactivity potential in multi-species studies. The rabbit monoclonal antibody (bsm-61567r-biotin) offers broader species reactivity, making it versatile for comparative studies across human, mouse, and rat models .

How can researchers verify the specificity of ROCK2 antibodies in their experimental systems?

To verify ROCK2 antibody specificity, researchers should implement a multi-layered validation approach:

• Knockout Controls: Use ROCK2 knockout cell lines (such as ROCK2 knockout HeLa cells) as negative controls in Western blots and other applications to confirm absence of signal where ROCK2 is not expressed .

• Peptide Competition Assays: Pre-incubate the antibody with the immunizing peptide/protein (for the rabbit polyclonal antibody, the recombinant human ROCK2 protein fragment spanning amino acids 1109-1388) before application to samples. Specific binding should be blocked by the competing antigen .

• Multiple Detection Methods: Confirm findings using alternative detection methods or multiple antibodies targeting different epitopes of ROCK2.

• Positive Control Cell Lines: Include known ROCK2-expressing cell lines (HepG2, A549) as positive controls to verify detection at the expected molecular weight (~175 kDa observed vs. 161 kDa predicted) .

This comprehensive validation strategy ensures that observed signals genuinely represent ROCK2 rather than non-specific binding or cross-reactivity with related proteins .

How can biotin-conjugated ROCK2 antibodies be utilized in studies of thermogenic programming in adipose tissue?

Recent research has revealed ROCK2's significant role in adipogenesis and thermogenic programming, making ROCK2 antibodies valuable tools in metabolism and obesity research. For investigating ROCK2's function in adipose tissue thermogenesis, researchers can:

• Use biotin-conjugated ROCK2 antibodies in Western blotting to quantify ROCK2 expression levels in different adipose tissue depots (white versus beige/brown) under various treatment conditions, including ROCK2 inhibition.

• Apply immunocytochemistry with biotin-conjugated ROCK2 antibodies to visualize ROCK2 localization in stromal-vascular (SV) cell cultures during differentiation into white versus beige adipocytes.

• Implement co-immunoprecipitation experiments with biotin-conjugated ROCK2 antibodies to identify interaction partners specific to thermogenic versus non-thermogenic adipocytes.

These applications are particularly relevant given findings that ROCK2 inhibition enhances beige adipogenesis with increased thermogenic gene expression, and that ROCK2 activity-mediated actin cytoskeleton dynamics inhibit beige adipogenesis in white adipose tissue (WAT). Furthermore, ROCK2 inhibition appears to counteract age-related and diet-induced fat mass gain and insulin resistance .

What methodological approaches can resolve contradictory findings between in vitro and in vivo ROCK2 studies?

To address discrepancies between in vitro and in vivo ROCK2 studies, particularly regarding adipogenesis, researchers should consider these methodological approaches:

• Tissue-Specific Conditional Knockouts: Generate adipose-specific or cell-type-specific ROCK2 knockout models to isolate tissue-specific effects from systemic compensation mechanisms.

• Knockin Point Mutations: Utilize ROCK2 knockin mice harboring kinase-dead (KD) point mutations (such as Lysine121 to Alanine substitution) to specifically examine kinase-dependent functions while preserving protein-protein interactions .

• Selective Pharmacological Inhibition: Apply specific ROCK2 inhibitors (like KD025) in both in vitro and in vivo systems to directly compare acute pharmacological inhibition versus genetic modification effects .

• Temporal Regulation: Implement inducible systems to control the timing of ROCK2 inhibition/depletion, distinguishing developmental versus acute metabolic effects.

• Quantitative Analysis: Employ biotin-conjugated ROCK2 antibodies in quantitative western blotting and ELISA to precisely measure ROCK2 expression and activation levels across different experimental paradigms.

Research has shown that ROCK2 deficient mouse embryonic fibroblasts (MEFs) exhibit enhanced adipogenesis in vitro, but similar phenotypes were not observed in partial ROCK2 deficient mice or transgenic mice expressing adipocyte-specific dominant-negative RhoA. These contradictions highlight the need for methodological refinement and careful experimental design .

How does post-translational modification affect ROCK2 antibody epitope recognition?

Post-translational modifications (PTMs) of ROCK2 can significantly impact antibody recognition through several mechanisms:

• Epitope Masking: Phosphorylation, ubiquitination, or other modifications may alter the three-dimensional structure of ROCK2, potentially obscuring epitopes targeted by specific antibodies.

• Conformational Changes: ROCK2 undergoes conformational changes upon activation that may expose or conceal certain epitopes, affecting antibody binding efficacy in active versus inactive states.

• Specific Domain Recognition: Some biotin-conjugated ROCK2 antibodies target specific domains or amino acid sequences (such as AA 1109-1388 in the polyclonal antibody ABIN7370535). PTMs within or adjacent to these regions can directly interfere with antibody binding .

To address these challenges, researchers should:

• Use multiple antibodies targeting different ROCK2 epitopes when studying PTM-rich contexts

• Consider phospho-specific antibodies when studying ROCK2 activation states

• Validate antibody performance under experimental conditions that may induce relevant PTMs

• Include appropriate controls treated with phosphatases or other enzymes that remove specific PTMs

These considerations are particularly important when studying ROCK2 in signaling contexts where its regulation via phosphorylation or other modifications is dynamically changing .

What are the common technical challenges when using biotin-conjugated ROCK2 antibodies in Western blotting?

When using biotin-conjugated ROCK2 antibodies for Western blotting, researchers frequently encounter these technical challenges and solutions:

• High Molecular Weight Detection: ROCK2 is a large protein (~161 kDa predicted, often observed at ~175 kDa due to post-translational modifications), requiring optimization of transfer conditions:

  • Use low percentage (6-8%) SDS-PAGE gels

  • Implement extended transfer times or semi-dry transfer systems

  • Consider specialized transfer buffers for high molecular weight proteins

• Endogenous Biotin Interference: Certain cell types contain high levels of endogenous biotin that can cause background:

  • Block with avidin/streptavidin before primary antibody incubation

  • Use specialized blocking reagents designed to block endogenous biotin

  • Consider alternative detection methods when working with biotin-rich tissues

• Optimizing Dilution: The recommended dilution range (1:300-5000) is broad:

  • Begin with a mid-range dilution (1:1000)

  • Perform titration experiments to determine optimal antibody concentration

  • Adjust based on ROCK2 expression levels in your specific samples

• Signal Development: When using streptavidin-HRP systems:

  • Optimize incubation time with streptavidin-HRP (typically 30-60 minutes)

  • Use freshly prepared ECL substrate

  • Consider signal enhancement systems for low abundance detection

• Non-specific Binding: High background can obscure specific signals:

  • Increase washing steps (5-6 washes recommended)

  • Optimize blocking conditions (5% BSA often works better than milk for phospho-proteins)

  • Consider using TBS-T with higher Tween-20 concentration (0.1-0.2%)

How can researchers optimize quantitative ELISA assays for ROCK2 using biotin-conjugated antibodies?

For optimal quantitative ELISA performance using biotin-conjugated ROCK2 antibodies, researchers should implement these methodological refinements:

• Standard Curve Optimization:

  • Use recombinant ROCK2 protein for standard curve preparation

  • Implement 7-8 point standard curves with 2-fold dilutions

  • Ensure the standard curve encompasses the expected concentration range of samples

  • Apply 4-parameter logistic regression for curve fitting

• Sample Preparation:

  • Optimize protein extraction buffers to efficiently solubilize ROCK2 while preserving epitope integrity

  • Standardize protein concentration across samples (BCA or Bradford assay)

  • Pre-clear samples with protein A/G beads to reduce non-specific binding

• Assay Protocol Refinements:

  • Maintain consistent 37°C incubation temperature throughout the protocol

  • Implement precisely timed 80-minute sample incubation

  • Maintain exactly 50-minute incubation for both biotinylated antibody and streptavidin-HRP steps

  • Ensure complete washing between steps (3-5 washes recommended)

  • Control TMB substrate development time (20 minutes in darkness) for reproducible results

• Controls:

  • Include blank, negative control, and positive control wells in each assay

  • Consider spike-recovery experiments to validate quantitative accuracy

  • Run technical replicates (minimum duplicates, preferably triplicates) for all samples and standards

• Data Analysis:

  • Subtract blank readings from all measurements

  • Apply curve fitting algorithms appropriate for immunoassays

  • Calculate intra-assay and inter-assay coefficients of variation to assess reliability

  • Consider normalization to total protein content for comparative analyses

What strategies can address low signal-to-noise ratios when working with biotin-conjugated ROCK2 antibodies?

To improve signal-to-noise ratios when working with biotin-conjugated ROCK2 antibodies, researchers should consider these strategic approaches:

• Blocking Optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Include carrier proteins (0.1-0.5% BSA) in antibody dilution buffers

• Antibody Quality:

  • Validate antibody specificity using knockout controls

  • Confirm antibody activity via dot blot before complex experiments

  • Consider antibody fragmentation (using Fab fragments) to reduce non-specific binding

• Enhanced Washing:

  • Increase wash buffer volume (use at least 200 μL per well in microplate formats)

  • Extend washing duration (30-60 seconds per wash)

  • Increase the number of wash steps (5-6 washes after primary and secondary antibody steps)

• Signal Amplification Systems:

  • Implement tyramide signal amplification for immunohistochemistry applications

  • Use poly-HRP conjugated streptavidin for enhanced sensitivity

  • Consider biotin-streptavidin multiplexing strategies for signal enhancement

• Detection System Optimization:

  • For colorimetric detection, optimize substrate development time

  • For fluorescence detection, use narrow bandpass filters to reduce background

  • For chemiluminescence, use high-sensitivity substrates and optimize exposure times

• Sample Preparation:

  • Implement additional purification steps to remove interfering substances

  • Use detergent-compatible protein extraction methods

  • Consider subcellular fractionation to enrich for ROCK2-containing fractions