Recombinant Crassostrea gigas A-kinase anchor protein 7 isoform X2 Antibody

What is A-kinase anchor protein 7 and what is its function in Crassostrea gigas?

A-kinase anchor protein 7 (AKAP7) belongs to a family of functionally related proteins that bind to regulatory subunits of cAMP-dependent protein kinase A (PKA), targeting the enzyme to specific subcellular compartments. In Crassostrea gigas (Pacific oyster), AKAP7 isoform X2 is believed to regulate signal transduction pathways similar to its mammalian counterparts. While mammalian AKAP7 targets PKA to plasma membranes and permits functional coupling to L-type calcium channels and affects epithelial sodium channel (ENaC) activity , the specific functions of C. gigas AKAP7 are still being characterized. Based on structural homology, it likely plays crucial roles in signaling complexes during development and physiological responses in this bivalve species.

What are the structural characteristics of C. gigas AKAP7 isoform X2?

Crassostrea gigas AKAP7 isoform X2 has the following characteristics:

• Amino acid sequence: MARRKRESPNYFLAIQITEDEIKRNVREIQEIILAKEENLSTAMIGIDTLHLTLGVYYLEDGFSIIQIKRALDKFHSQLKAADFVPPCLKVSTLGHFNHKVLYASLEENQGLEELNTLVNGVRTSLENDGVFTADDRYTPHVTISKMSKDMNRLRKLGVSRIDPSHYQEKRTAYFGQQVVKSIQLCAMNVPKTESGYYYVEHEIMFS

• Expression region: 1-207aa

• Molecular weight: 25.8 kDa

• UniProt ID: XP_011412823.1

What is the tissue expression pattern of AKAP7 in C. gigas?

While the search results don't provide specific information about AKAP7 expression patterns in C. gigas, we can extrapolate from related studies. In mollusks, signaling proteins often show tissue-specific expression patterns. For instance, the retinoic acid receptor (CgRAR) in C. gigas has been shown to have higher expression levels in hemocytes and gonads , suggesting important roles in immune response and reproduction.

A comprehensive tissue expression analysis for C. gigas AKAP7 would likely include:

Note: Specific expression data for C. gigas AKAP7 would require tissue-specific RT-qPCR analysis similar to methods used for other C. gigas proteins .

What are the optimal storage and handling conditions for recombinant C. gigas AKAP7 antibodies?

Based on recommendations for similar recombinant proteins:

• Storage temperature: Store at -20°C/-80°C for long-term storage

• Working aliquots: Store at 4°C for up to one week

• Shelf life: Approximately 6 months for liquid form at -20°C/-80°C; 12 months for lyophilized form

• Avoid: Repeated freeze-thaw cycles

• Buffer considerations: For long-term storage, consider adding a carrier protein (0.1% HSA or BSA)

Proper aliquoting upon first thaw is strongly recommended to avoid protein degradation from repeated freeze-thaw cycles. When designing experiments, researchers should validate the integrity of the antibody by Western blot before use in critical experiments.

What validation methods should be used to confirm antibody specificity for C. gigas AKAP7?

To validate antibody specificity for C. gigas AKAP7 isoform X2, researchers should:

• Western blot analysis: Confirm a single band at the expected molecular weight (25.8 kDa plus tag size)

• Immunoprecipitation followed by mass spectrometry: Verify that the precipitated protein is indeed C. gigas AKAP7

• Peptide competition assay: Pre-incubate antibody with purified AKAP7 peptide before immunostaining to confirm signal reduction

• Cross-reactivity testing: Test against other AKAP family members, particularly other C. gigas AKAPs

• Knockout or knockdown controls: If available, use AKAP7-deficient samples as negative controls

A comprehensive validation approach might include:

What expression systems are most effective for producing functional C. gigas AKAP7?

• E. coli expression system:

  • Advantages: High yield, cost-effective, simple culturing conditions

  • Limitations: Lack of post-translational modifications, potential for inclusion body formation

  • Recommended for: Basic structural studies, antibody production, interaction studies

• Insect cell expression system:

  • Advantages: Better folding, some post-translational modifications

  • Limitations: More expensive, technically demanding

  • Recommended for: Functional studies requiring properly folded protein

• Mammalian cell expression system:

  • Advantages: Native-like post-translational modifications, proper folding

  • Limitations: Lower yield, highest cost

  • Recommended for: Studies of protein function in cellular context, complex formation studies

For most basic research applications with C. gigas AKAP7, the E. coli system with a C-terminal 6xHis-tag has proven sufficient .

How can researchers effectively study protein-protein interactions involving C. gigas AKAP7?

To study protein-protein interactions involving C. gigas AKAP7, researchers can employ several complementary techniques:

• Co-immunoprecipitation (Co-IP): Similar to the methodology used in AKAP220 studies , researchers can use antibodies against C. gigas AKAP7 to precipitate protein complexes from oyster tissue lysates or heterologous expression systems.

• Surface Plasmon Resonance (SPR): This technique can determine binding kinetics between AKAP7 and potential partners, similar to methods used for AKAP7γ dimerization studies .

• Pulldown assays with recombinant proteins: Using His-tagged or MBP-tagged C. gigas AKAP7 to identify binding partners from tissue lysates.

• Proximity Ligation Assay (PLA): For detecting protein-protein interactions in situ in C. gigas tissues.

What role might AKAP7 play in Pacific oyster development or immune responses?

Given the important role of AKAPs in organizing signaling complexes, C. gigas AKAP7 may be involved in several physiological processes:

• Development and metamorphosis: C. gigas undergoes complex metamorphosis from pelagic larvae to settled juveniles. Studies have shown that larval settlement and adhesion involve specific protein secretion . AKAP7 might participate in signaling cascades during these developmental transitions.

• Immune response: The expression of various signaling molecules in hemocytes suggests roles in immune function. AKAP7 could organize signaling complexes involved in pathogen response.

• Environmental adaptation: As filter feeders in variable environments, oysters require sophisticated signal transduction networks for adaptation. AKAP7 may help coordinate cellular responses to environmental stressors.

• Reproductive development: Given the high expression of some signaling proteins in gonads , AKAP7 might participate in gametogenesis or reproductive cycles.

Experimental approaches to investigate these roles might include:

• Temporal expression analysis during development

• Expression changes in response to immune challenges

• Localization studies in various tissues under different conditions

What are common technical challenges when working with C. gigas AKAP7 antibodies?

Researchers working with C. gigas AKAP7 antibodies might encounter several challenges:

• Cross-reactivity issues: Due to conservation among AKAP family members, antibodies may recognize multiple proteins. Validation using appropriate controls is essential.

• Limited tissue specificity: The antibody may show different affinities across various C. gigas tissues due to post-translational modifications or protein complex formation.

• Fixation sensitivity: For immunohistochemistry applications, certain fixatives may mask epitopes. Comparing multiple fixation protocols is recommended:

• Sample preparation challenges: Marine invertebrate tissues often contain compounds that can interfere with immunological applications. Thorough washing and blocking steps are crucial.

• Degradation during extraction: Proteases in oyster tissues may degrade AKAP7 during extraction. Use of multiple protease inhibitors is recommended.

How can researchers optimize immunolocalization of AKAP7 in C. gigas tissues?

For optimal immunolocalization of AKAP7 in C. gigas tissues:

• Tissue preparation:

  • Fix fresh tissues immediately after collection

  • Consider testing both cryosectioning and paraffin embedding

  • For larvae and juvenile specimens, whole-mount immunostaining may be effective

• Antigen retrieval:

  • For paraffin sections, citrate buffer (pH 6.0) heat-mediated antigen retrieval

  • For difficult epitopes, try enzymatic retrieval with proteinase K

• Blocking and permeabilization:

  • Extended blocking (2-3 hours) with normal serum (5-10%) and BSA (3-5%)

  • Add 0.2-0.3% Triton X-100 for adequate permeabilization

  • Consider adding 0.1% Tween-20 to all wash buffers

• Antibody incubation:

  • Test a range of concentrations (1:100 to 1:1000)

  • Extended incubation at 4°C (overnight to 48 hours)

  • Include appropriate controls (pre-immune serum, secondary antibody only)

• Signal detection:

  • Consider tyramide signal amplification for low abundance proteins

  • Use confocal microscopy to accurately determine subcellular localization

  • Counter-stain with DAPI and phalloidin to provide cellular context

How can C. gigas AKAP7 be used in comparative studies of signaling pathway evolution?

C. gigas AKAP7 offers a valuable tool for evolutionary studies of signaling pathways:

• Phylogenetic analysis: Comparing AKAP7 sequences across mollusks, other invertebrates, and vertebrates can reveal evolutionary conservation of signaling scaffolds. Researchers could construct phylogenetic trees using maximum likelihood methods with programs like MEGA or RAxML.

• Functional domain conservation: Analysis of conserved domains between C. gigas and other species can reveal which functional aspects have been maintained throughout evolution:

  • PKA-binding domains

  • Membrane-targeting domains

  • Protein-protein interaction motifs

• Interactome comparison: Identifying C. gigas AKAP7 binding partners and comparing them with mammalian AKAP7 interactomes can reveal conservation of signaling networks.

• Heterologous expression studies: Expressing C. gigas AKAP7 in mammalian cells and assessing its ability to interact with mammalian PKA and other signaling proteins could reveal functional conservation.

• Ancestral state reconstruction: Using computational approaches to infer the ancestral state of AKAP proteins could provide insights into the evolution of compartmentalized signaling.

What emerging technologies could advance research on C. gigas AKAP7?

Several emerging technologies could significantly advance C. gigas AKAP7 research:

• CRISPR/Cas9 genome editing in C. gigas:

  • Creating AKAP7 knockout or tagged lines

  • Introducing mutations to study structure-function relationships

  • Enabling live tracking of AKAP7 with fluorescent tags

• Proximity labeling techniques:

  • BioID or TurboID fused to AKAP7 to identify proximal proteins in native contexts

  • APEX2 for electron microscopy visualization of AKAP7 complexes at ultrastructural level

• Single-cell transcriptomics and proteomics:

  • Analyzing AKAP7 expression in different cell types within C. gigas tissues

  • Correlating AKAP7 expression with cell-specific signaling networks

• Cryo-electron microscopy:

  • Determining the structure of C. gigas AKAP7 complexes

  • Comparing with mammalian AKAP structures to reveal evolutionary changes

• Optogenetic approaches:

  • Creating light-sensitive AKAP7 variants to control PKA localization

  • Studying real-time effects of disrupting AKAP7-mediated signaling

How might understanding C. gigas AKAP7 contribute to comparative signal transduction research?

Research on C. gigas AKAP7 can provide valuable insights into evolutionary aspects of compartmentalized signaling:

• Divergent mechanisms: Identifying unique aspects of molluscan AKAP signaling could reveal alternative mechanisms for achieving similar cellular outcomes. For example, while mammalian AKAP7 regulates L-type calcium channels and sodium channels , C. gigas AKAP7 might organize different effector proteins.

• Environmental adaptation insights: Understanding how signaling scaffolds function in organisms adapted to variable marine environments could reveal mechanisms of cellular resilience.

• Signaling network evolution: Comparative studies between C. gigas and model organisms could reveal how complexity in signaling networks evolved. Did AKAPs acquire new functions as organisms became more complex, or did they maintain core functions while adapting to new cellular contexts?

• Developmental biology perspectives: Studying AKAP7 in C. gigas larval development could provide insights into the role of compartmentalized signaling during metamorphosis and settlement, processes that have been lost in many model organisms.

• Evolutionary medicine applications: Understanding fundamental differences in signaling mechanisms between invertebrates and vertebrates could potentially reveal new therapeutic targets that selectively affect vertebrate pathways.