SPAC1565.02c Antibody

What is SPAC1565.02c and what is its biological function?

SPAC1565.02c is a gene encoding a putative Rho GTPase-activating protein (RhoGAP) in Schizosaccharomyces pombe. This protein is predicted to function as a regulator of Rho-type GTPases, which act as molecular switches controlling various cellular processes. As a RhoGAP, SPAC1565.02c likely catalyzes the hydrolysis of GTP bound to active Rho GTPases, thereby inactivating them and regulating downstream signaling pathways involved in cytoskeletal organization, cell morphology, and other cellular functions . The protein contains a BCH (BNIP-2 and Cdc42GAP Homology) domain, which is known to target small GTPases and their regulators, making it a critical component in cellular signaling networks.

What are the key characteristics of antibodies against SPAC1565.02c?

Commercially available SPAC1565.02c antibodies are typically rabbit polyclonal antibodies raised against Schizosaccharomyces pombe (strain 972/24843). These antibodies are purified through antigen-affinity methods and belong to the IgG isotype . They are designed to specifically recognize and bind to SPAC1565.02c protein, enabling researchers to detect and study this protein in various experimental contexts. The antibodies are validated for applications such as ELISA and Western Blot analysis, making them versatile tools for investigating SPAC1565.02c expression, localization, and interactions with other proteins.

How is the SPAC1565.02c antibody related to human RhoGAP research?

While SPAC1565.02c is a yeast protein, it shares significant homology with human p50RhoGAP (ARHGAP1), particularly in the BCH domain region. Sequence alignment reveals approximately 46% sequence similarity between the BCH domains of S. pombe SPAC1565.02c and human p50RhoGAP, including conservation of a BCH domain signature motif: R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs (where "h" refers to large and hydrophobic residues and "s" refers to small and weakly polar residues) . This homology makes SPAC1565.02c antibodies valuable tools for comparative studies of RhoGAP function across species and for investigating evolutionarily conserved mechanisms of Rho GTPase regulation.

What are the optimal conditions for using SPAC1565.02c antibody in Western Blot applications?

For Western Blot applications using SPAC1565.02c antibody, researchers should consider the following protocol:

• Sample preparation: Extract proteins from S. pombe cells using a lysis buffer containing protease inhibitors to prevent protein degradation.

• Protein separation: Separate proteins by SDS-PAGE using a 10-12% gel, which is optimal for the molecular weight range of SPAC1565.02c.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard protocols.

• Blocking: Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute SPAC1565.02c antibody to the manufacturer's recommended concentration (typically 1:500 to 1:2000) in blocking solution and incubate overnight at 4°C.

• Washing: Wash the membrane 3-4 times with TBST to remove unbound antibody.

• Secondary antibody incubation: Incubate with an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) for 1-2 hours at room temperature.

• Detection: Visualize using ECL substrate and appropriate imaging equipment.

It's critical to optimize antibody concentration for your specific experimental conditions, as signal-to-noise ratio can vary based on protein expression levels and sample preparation methods .

How can SPAC1565.02c antibody be used to study RhoGAP domain function and regulation?

To study RhoGAP domain function and regulation using SPAC1565.02c antibody, researchers can employ several approaches:

• Co-immunoprecipitation (Co-IP): Use the antibody to pull down SPAC1565.02c and identify interacting proteins, particularly Rho GTPases and other signaling molecules.

• GAP activity assays: After immunoprecipitation with SPAC1565.02c antibody, the captured protein can be used in in vitro GTPase activity assays to measure its ability to stimulate GTP hydrolysis by Rho GTPases.

• Domain mapping studies: Combined with site-directed mutagenesis of key residues in the BCH or GAP domains, the antibody can be used to detect how mutations affect protein stability, localization, or function.

• Autoinhibition analysis: Since the BCH domain can autoinhibit the GAP domain in homologous proteins like p50RhoGAP, the antibody can be used to study how various conditions or mutations (particularly in the β5-strand) affect this regulatory mechanism .

These approaches allow researchers to dissect the molecular mechanisms underlying SPAC1565.02c function and its role in regulating Rho GTPase signaling.

How can structural insights from the BCH domain of SPAC1565.02c inform functional studies?

The crystal structure of the BCH domain from SPAC1565.02c reveals an intertwined dimeric architecture with asymmetric monomers, containing both a unique RhoA-binding loop and a lipid-binding pocket . These structural features provide several avenues for functional investigation:

These structure-guided functional studies can significantly advance our understanding of how SPAC1565.02c and related RhoGAPs regulate GTPase signaling pathways .

What techniques can be used to study the interaction between SPAC1565.02c's BCH domain and its GAP domain?

To investigate the interaction between the BCH and GAP domains of SPAC1565.02c, researchers can employ several sophisticated techniques:

• Förster Resonance Energy Transfer (FRET): By tagging the BCH and GAP domains with appropriate fluorophores, researchers can measure domain interaction in real-time in living cells.

• Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can identify regions of the protein that become protected from exchange upon domain interaction, providing a map of the interaction interface.

• Nuclear Magnetic Resonance (NMR) spectroscopy: For studying dynamic interactions between domains in solution.

• Cross-linking coupled with mass spectrometry: To identify specific residues involved in domain-domain contacts.

• Antibody-based approaches: The SPAC1565.02c antibody can be used in proximity ligation assays (PLA) to visualize domain interactions in situ, or in pull-down assays with truncated constructs to map interaction regions.

These methods, when used in combination with site-directed mutagenesis targeting the β5-strand region, can provide comprehensive insights into the molecular mechanisms of autoinhibition and activation .

How should researchers interpret contradictory results when studying SPAC1565.02c activity across different experimental systems?

When faced with contradictory results across experimental systems, researchers should consider several potential explanations:

• Expression levels: Different expression systems may produce varying levels of SPAC1565.02c, potentially affecting its dimerization, localization, or activity. Western blot quantification using the antibody can help normalize for expression differences.

• Post-translational modifications: Variations in post-translational modifications across systems may affect protein function. Phosphorylation-specific antibodies or mass spectrometry analyses can identify such differences.

• Interactome variations: The repertoire of interacting proteins may differ between systems, affecting SPAC1565.02c function. Co-IP experiments using the antibody can identify system-specific binding partners.

• Lipid environment differences: Since the BCH domain has a lipid-binding pocket, variations in membrane composition between systems could affect function .

• Experimental conditions: Differences in buffer conditions, temperature, or other experimental parameters may influence protein activity.

To address contradictions, researchers should:

• Perform careful controls with the antibody to verify protein integrity and expression

• Test multiple experimental approaches to confirm findings

• Consider using both in vitro and in vivo systems to comprehensively characterize protein function

• Document all experimental conditions thoroughly to identify variables that might explain discrepancies

What are the potential sources of false positive/negative results when using SPAC1565.02c antibody, and how can they be mitigated?

Regular validation of antibody specificity using appropriate positive and negative controls is essential for reliable research results. When possible, complement antibody-based techniques with orthogonal methods that don't rely on antibody recognition .

How does the dimeric structure of the BCH domain influence SPAC1565.02c function?

The intertwined dimeric structure of the BCH domain revealed in crystallographic studies has significant implications for SPAC1565.02c function:

• Cooperative regulation: The asymmetric nature of the dimer suggests that binding events at one monomer might allosterically affect the other, potentially allowing for cooperative regulation of GAP activity.

• Multivalent interactions: Dimerization creates a scaffold with multiple binding surfaces, potentially allowing simultaneous interaction with Rho GTPases, lipids, and other signaling molecules.

• Spatial coordination: The dimeric structure may facilitate the coordination of multiple Rho GTPases in close proximity, enabling efficient regulation of localized GTPase activity.

• Stability and regulation: The intermolecular β-sheet involving the β5-strand of the BCH domain plays a crucial role in dimer stability and in the autoinhibition of the adjacent GAP domain. Mutations destabilizing this structure can release autoinhibition, rendering the GAP domain active and leading to enhanced RhoA inactivation .

• Self-association: When the autoinhibitory interaction between the BCH and GAP domains is disrupted, the released BCH domains show enhanced protein-protein interaction, potentially leading to higher-order oligomerization.

These structural features provide a mechanistic basis for understanding how SPAC1565.02c and related RhoGAPs achieve precise spatiotemporal regulation of Rho GTPase signaling .

What is the significance of the lipid-binding pocket in the BCH domain for SPAC1565.02c function?

The lipid-binding pocket identified in the BCH domain of SPAC1565.02c has important functional implications:

• Membrane localization: The lipid-binding capability likely helps localize SPAC1565.02c to specific membrane compartments where its target GTPases function.

• Prenylated GTPase recognition: The pocket appears specialized to accommodate the prenylation tail of Rho GTPases, which are post-translationally modified with lipid moieties. This interaction may be critical for proper substrate recognition and GAP activity .

• Specificity determination: The characteristics of the lipid-binding pocket may contribute to the specificity of SPAC1565.02c for certain Rho family GTPases over others.

• Regulatory mechanism: Lipid binding might induce conformational changes that affect the autoinhibitory interaction between the BCH and GAP domains.

• Therapeutic targeting: Understanding the structure and function of this lipid-binding pocket could inform the development of small molecules that modulate RhoGAP activity in research or therapeutic contexts.

Research has shown that a lipid moiety on Rho GTPases is necessary for their efficient inactivation by related RhoGAPs, supporting the functional importance of this lipid-binding region . Mutations affecting the lipid-binding pocket could provide valuable insights into its role in SPAC1565.02c function and Rho GTPase regulation.