AKAP7 Antibody

What is AKAP7 and why is it significant in cellular signaling research?

AKAP7, also known as AKAP15 or AKAP18, is a member of the A-kinase anchoring protein family that binds to regulatory subunits of cAMP-dependent protein kinase A (PKA) and targets the enzyme to specific subcellular compartments. AKAP7 plays crucial roles in cellular signaling by localizing PKA in complexes with various proteins including calcium channels (CaV1.2) and phospholamban (PLN) . It's particularly significant in research involving cardiac calcium dynamics, neuronal function, and ion channel regulation . AKAP7 exists in multiple splice variants (α, β, γ, and δ isoforms) with different subcellular localizations and functions, making it an important target for studying compartmentalized signaling pathways .

Which AKAP7 isoforms can be detected by commercially available antibodies?

Most commercial antibodies, such as the 12591-1-AP polyclonal antibody, can recognize all isoforms of AKAP7 . This comprehensive detection is possible because many antibodies are raised against conserved regions or use immunogens that contain sequences common to multiple isoforms. For example, the antibody described in search result #7 uses an immunogen sequence corresponding to amino acids 1-81 encoded by BC016927, which allows it to detect multiple AKAP7 isoforms . When selecting an antibody for specific isoform detection, researchers should carefully review the immunogen information and validation data provided by manufacturers.

What are the molecular weights of different AKAP7 isoforms I should expect to see in Western blots?

When performing Western blots for AKAP7, you should expect to observe different molecular weight bands corresponding to specific isoforms:

It's important to note that the observed molecular weights may differ from calculated weights due to post-translational modifications or the presence of fusion tags in recombinant proteins . In some studies, AKAP7α has been detected as a 15-kDa protein in brain lysates, highlighting potential variability in observed weights across different experimental conditions .

What are the validated applications for AKAP7 antibodies, and what are the recommended dilutions for each technique?

Based on the search results, AKAP7 antibodies have been validated for multiple applications:

It's recommended to optimize these dilutions for your specific experimental conditions and sample types . For antigen retrieval in IHC, TE buffer at pH 9.0 is suggested, although citrate buffer at pH 6.0 can be used as an alternative .

How should I prepare tissue samples for optimal AKAP7 detection using IHC or IF techniques?

For optimal detection of AKAP7 in tissue samples using IHC or IF techniques:

• Fixation: Standard formalin fixation and paraffin embedding (FFPE) procedures are suitable for AKAP7 detection.

• Antigen Retrieval: For IHC, it's recommended to use TE buffer at pH 9.0 for antigen retrieval. Alternatively, citrate buffer at pH 6.0 can be used if needed .

• Blocking: Use 5% BSA in TBS-T (25 mM Tris-Cl pH 7.6, 150 mM NaCl, 0.1% Tween 20) for blocking non-specific binding sites .

• Primary Antibody Incubation: For IHC, dilute antibodies in the range of 1:20-1:200; for IF, use 1:10-1:100 dilutions. Incubate at 4°C overnight for optimal results .

• Washing: Perform five wash steps with TBS-T, each for 5 minutes, to reduce background .

• Detection Systems: For IHC, HRP-conjugated secondary antibodies with appropriate substrates can be used. For IF, fluorescently-labeled secondary antibodies appropriate for your microscopy setup should be selected .

When studying AKAP7 in tissue sections, note that AKAP7α is primarily expressed in brain and lung tissues, with very low expression in heart, while the long isoforms (AKAP7γ/δ) are more ubiquitously expressed and may require different optimization strategies .

What controls should be included when using AKAP7 antibodies to ensure experimental validity?

To ensure experimental validity when using AKAP7 antibodies, include the following controls:

• Positive Controls: Use tissues or cell lines known to express AKAP7, such as:

  • Human brain tissue for AKAP7α detection

  • HepG2 cells for immunofluorescence studies

  • Multiple tissues for long isoforms (AKAP7γ/δ)

• Negative Controls:

  • AKAP7 knockout tissue/cells when available - research has developed AKAP7 KO mice where all isoforms have been deleted

  • Primary antibody omission control

  • Isotype control using non-specific IgG of the same host species

• Loading Controls:

  • For Western blots, include housekeeping proteins such as β-actin, GAPDH, or another stable reference protein

  • For normalization of phosphorylation studies, probing for total protein alongside phospho-specific antibodies is essential

• Expression Validation:

  • Consider complementary methods like RT-PCR to confirm expression patterns, especially when distinguishing between isoforms

  • When studying tissue-specific expression, quantitative RT-PCR can serve as a more sensitive detection method than Western blotting alone

As demonstrated in the study by Jones et al. (2012), proper controls were crucial in validating their findings regarding the role of AKAP7 in cardiomyocytes, including the use of knockout mice and appropriate normalization to housekeeping proteins .

How can AKAP7 antibodies be effectively used to study protein-protein interactions?

AKAP7 antibodies can be effectively used to study protein-protein interactions through several methods:

• Co-Immunoprecipitation (Co-IP):

  • AKAP7 antibodies can precipitate AKAP7 and its interacting partners from cell or tissue lysates

  • As demonstrated in published studies, Co-IP can be performed using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

  • The precipitated complexes can then be analyzed by Western blotting to identify interacting proteins

• Pull-down Assays:

  • As described in the study by Whiting et al. (2015), purified His/S-tagged AKAP7γ can be incubated with potential binding partners such as AKAP7γ-MBP

  • The complexes can be isolated using appropriate resins (e.g., amylose resin for MBP-tagged proteins)

  • After washing, the components of the complex can be separated by SDS-PAGE and analyzed by Western blot

• Peptide Arrays:

  • AKAP7 binding sites can be mapped using peptide arrays

  • As described in the literature, 20-mer peptides with three amino acid offsets can be synthesized on membranes

  • The membranes can be incubated with recombinant S-tagged AKAP7γ and binding detected using HRP-conjugated anti-S-tag antibodies

• Proximity Ligation Assays:

  • For in situ detection of protein interactions in intact cells or tissues

  • Requires AKAP7 antibodies from different host species or directly conjugated antibodies

These methods have been successfully used to investigate interactions between AKAP7 and its binding partners, including dimerization of AKAP7γ and interactions with ion channels and regulatory proteins .

How do AKAP7 expression patterns differ across tissues, and what methodological considerations should researchers address when studying tissue-specific AKAP7 functions?

AKAP7 expression patterns vary significantly across tissues, with important implications for research design:

Tissue Expression Patterns:

Methodological Considerations:

• Detection Sensitivity:

  • For tissues with low AKAP7 expression, consider using immunoprecipitation to concentrate and enrich the protein before detection

  • Quantitative RT-PCR provides a more sensitive method to detect expression levels, especially for distinguishing between short and long isoforms

• Species Differences:

  • Expression patterns vary between species - for example, AKAP7β shows conserved expression in kidney between rodents and humans, but has predominant expression in pancreas in humans

  • Validate antibodies in your specific species of interest, as cross-reactivity may vary

• Subcellular Localization:

  • In neurons, AKAP7α localizes to both dendrites and mossy fiber axonal projections

  • Consider subcellular fractionation or high-resolution imaging methods to accurately determine localization

• Functional Studies:

  • For tissue-specific functions, knockout models targeting specific tissues (e.g., dentate granule cells for AKAP7 in hippocampus) provide valuable insights

  • Consider the impact of different isoforms in specific tissues - for example, AKAP7α in dentate granule cells plays a role in spatial discrimination

As demonstrated in the study by Jones et al. (2012), using multiple complementary methods (Western blotting, immunoprecipitation, and quantitative RT-PCR) provides a more complete picture of AKAP7 expression across tissues .

What are the challenges in studying AKAP7 phosphorylation-dependent signaling, and how can antibody-based approaches help overcome them?

Studying AKAP7 phosphorylation-dependent signaling presents several challenges that can be addressed through strategic antibody-based approaches:

Challenges:

• Isoform Specificity: Different AKAP7 isoforms may participate in distinct signaling pathways and be regulated differently by phosphorylation events.

• PKA Co-localization Dynamics: AKAP7 anchors PKA to specific subcellular compartments, making it challenging to study the spatial and temporal dynamics of PKA-dependent phosphorylation events.

• Substrate Identification: Identifying specific substrates of AKAP7-anchored PKA in different cell types and compartments is complex.

• Signal Integration: AKAP7 may integrate multiple signaling pathways, requiring simultaneous detection of different phosphorylation events.

Antibody-Based Solutions:

• Phospho-Specific Antibodies for Substrates:

  • Use antibodies that specifically recognize phosphorylated forms of known AKAP7-associated substrates, such as phospho-PLN (Ser16) or phospho-CaV1.2 (Ser1928)

  • These can be used in Western blotting to quantify phosphorylation levels in response to different stimuli

• Multiplexed Phosphorylation Analysis:

  • Employ antibodies against total and phosphorylated forms of multiple AKAP7-associated proteins simultaneously

  • For example, in the study by Jones et al. (2012), researchers simultaneously analyzed phosphorylation of CaV1.2 and PLN in response to β-adrenergic stimulation

• Proximity Ligation Assays:

  • Use antibodies against AKAP7 and phosphorylated substrates in proximity ligation assays to detect close associations between AKAP7 and its phosphorylated targets in situ

• Pharmacological Manipulation:

  • Combine antibody detection with pharmacological tools that modulate PKA activity, such as isoproterenol (β-adrenergic agonist), to study dynamic phosphorylation events

  • Antibodies can then be used to detect changes in phosphorylation status following treatment

• Comparative Analysis in Knockout Models:

  • Use AKAP7 knockout models to compare phosphorylation patterns of putative substrates with and without AKAP7, helping to identify AKAP7-dependent phosphorylation events

  • This approach was effectively used in the study of cardiomyocytes from AKAP7 knockout mice, where phosphorylation of CaV1.2 and PLN was examined following β-adrenergic stimulation

By combining these antibody-based approaches with functional assays and genetic models, researchers can gain deeper insights into the complex phosphorylation-dependent signaling networks regulated by AKAP7.

How should researchers interpret Western blot results when detecting multiple bands with AKAP7 antibodies?

When multiple bands appear in Western blots using AKAP7 antibodies, systematic interpretation is crucial:

• Expected Multiple Isoforms:

  • AKAP7 antibodies may detect multiple isoforms simultaneously, resulting in bands at different molecular weights

  • The observed molecular weights for AKAP7 isoforms are: α (~11 kDa), β (~18 kDa), and γ/δ (~37 kDa)

  • Verify these patterns against positive control samples with known AKAP7 isoform expression

• Tissue-Specific Expression Patterns:

  • Consider tissue source when interpreting bands - AKAP7α is highly expressed in brain but may be barely detectable in other tissues

  • Long isoforms (γ/δ) are more ubiquitously expressed across tissues

  • Use the tissue expression data as a reference point for expected isoform patterns:

• Post-Translational Modifications:

  • Additional bands may represent post-translationally modified forms of AKAP7

  • Phosphorylation states may affect protein migration

  • Consider using phosphatase treatment to determine if higher molecular weight bands are due to phosphorylation

• Degradation Products:

  • Lower molecular weight bands might represent degradation products

  • Ensure proper sample preparation with protease inhibitors

  • Compare fresh samples with stored samples to assess degradation

• Non-Specific Binding:

  • Bands at unexpected molecular weights may represent non-specific binding

  • Include appropriate controls, especially AKAP7 knockout samples when available

  • Perform blocking optimization to reduce non-specific binding

If uncertain about band identity, consider complementary approaches such as RT-PCR to confirm the expression of specific isoforms in your samples, as demonstrated in the study by Jones et al. where they used both protein detection methods and mRNA analysis to confirm isoform expression patterns .

What factors can affect AKAP7 antibody specificity, and how can these be addressed in experimental design?

Several factors can affect AKAP7 antibody specificity, and researchers should address these through careful experimental design:

• Cross-reactivity with Related Proteins:

  • AKAP7 belongs to a family of structurally related AKAPs

  • For validation, compare staining patterns with multiple antibodies targeting different AKAP7 epitopes

  • Include AKAP7 knockout samples as negative controls when available

• Epitope Accessibility:

  • Protein folding, complex formation, or post-translational modifications may mask epitopes

  • For fixed tissues or cells, optimize antigen retrieval methods - TE buffer at pH 9.0 is recommended for AKAP7, with citrate buffer at pH 6.0 as an alternative

  • For native protein detection, try different detergents or lysis conditions that may better preserve or expose epitopes

• Fixation Effects:

  • Different fixation methods can affect epitope preservation

  • Consider comparing paraformaldehyde fixation with methanol fixation for immunofluorescence applications

  • Optimize fixation time based on tissue type and thickness

• Antibody Quality and Storage:

  • Use antibodies from reputable sources with validation data

  • Store according to manufacturer recommendations - typically at -20°C with 50% glycerol

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

• Background Reduction Strategies:

  • Optimize blocking conditions (5% BSA in TBS-T is commonly used)

  • Increase washing steps - five washes of 5 minutes each in TBS-T has been effective in published protocols

  • Consider using more dilute antibody with longer incubation times

  • For tissues with high endogenous peroxidase activity, include appropriate quenching steps before antibody incubation

• Validation Approaches:

  • Peptide competition assays where the antibody is pre-incubated with the immunizing peptide

  • RNA interference to reduce AKAP7 expression and confirm specificity

  • Use of multiple antibodies targeting different regions of AKAP7

  • Correlation of protein detection with mRNA expression using RT-PCR

By systematically addressing these factors, researchers can enhance the specificity and reliability of their AKAP7 antibody-based experiments, as demonstrated in published studies that employed multiple validation approaches .

How should data from AKAP7 knockout models be interpreted when evaluating antibody specificity and AKAP7 function?

Data from AKAP7 knockout models provides crucial information for both antibody validation and functional studies:

For Antibody Specificity Assessment:

• Complete Signal Loss:

  • In a true AKAP7 knockout, all specific antibody signals should be absent

  • As demonstrated in the study by Jones et al., Western blots from AKAP7 KO mice showed complete loss of both long and short isoforms of AKAP7

  • Similarly, immunohistochemistry showed specific expression of AKAP7 in wild-type dentate granule cells and complete loss in the KO

• Persistent Signals:

  • Any remaining signals in knockout tissues suggest:
    a) Non-specific binding of the antibody
    b) Incomplete knockout (check the knockout strategy - e.g., the study by Jones et al. used Cre/loxP recombination to delete the only common exon)
    c) Potential cross-reactivity with other AKAP family members

• Background Assessment:

  • Knockout tissues allow determination of the true background level for each application

  • This information can guide optimization of antibody dilutions and detection protocols

For Functional Interpretation:

When interpreting knockout data, it's important to consider potential differences between acute knockdown and constitutive knockout, where developmental compensation might occur. Additionally, species differences should be considered when translating findings between animal models and human studies .

How has our understanding of AKAP7 biology evolved, and what are emerging applications for AKAP7 antibodies in cutting-edge research?

Our understanding of AKAP7 biology has evolved significantly, opening new applications for AKAP7 antibodies:

Evolution of AKAP7 Understanding:

• Isoform Complexity:

  • Originally identified as a simple anchoring protein, AKAP7 is now known to exist in multiple splice variants with distinct subcellular localizations and functions

  • Molecular evolution studies have revealed the genesis of the AKAP7 RI/RII binding domain and evolutionary conservation of key protein regions in short variants, with more rapid change in long form variants

• Tissue-Specific Roles:

  • Initial focus on cardiac function has expanded to include roles in neuronal function, particularly in spatial discrimination and memory formation

  • AKAP7's involvement in long QT syndrome suggests roles in cardiac pathophysiology

• Functional Redundancy and Specificity:

  • Studies using knockout models revealed unexpected functional redundancy in certain tissues (e.g., cardiomyocytes), while showing essential roles in others (e.g., dentate granule cells)

Emerging Applications for AKAP7 Antibodies:

• Neuroscience Applications:

  • Investigation of AKAP7's role in synaptic plasticity using antibodies to track localization in neuronal subcellular compartments

  • Studies of AKAP7 in mossy fiber projections related to spatial memory formation

  • Potential applications in studying neurological disorders with memory deficits

• Cardiac Electrophysiology:

  • Examination of AKAP7's role in cardiac ion channel regulation and arrhythmias

  • Investigation of AKAP7's involvement in Long QT Syndrome 1, as indicated by GeneCards data

  • Studies of compensatory mechanisms in AKAP7-deficient cardiac tissues

• Single-Cell and Subcellular Resolution Studies:

  • Super-resolution microscopy combined with AKAP7 antibodies to study nanoscale organization of signaling complexes

  • Single-cell proteomics to examine AKAP7 expression heterogeneity within tissues

• Integrative Multi-omics Approaches:

  • Combination of AKAP7 antibody-based proteomics with transcriptomics and phosphoproteomics

  • Studies correlating AKAP7 expression/localization with phosphorylation patterns of downstream targets

• Translational Research:

  • Investigation of AKAP7's role in human disease contexts

  • Development of diagnostic or prognostic applications based on AKAP7 expression or localization patterns

  • Potential therapeutic targeting of AKAP7-dependent signaling pathways

As research tools and methodologies continue to advance, AKAP7 antibodies will remain essential for deciphering the complex roles of this multifunctional anchoring protein in normal physiology and disease states.

What methodological advances are improving the specificity and utility of AKAP7 antibodies in complex biological systems?

Recent methodological advances have significantly enhanced the specificity and utility of AKAP7 antibodies:

• Enhanced Validation Strategies:

  • Knockout validation using CRISPR/Cas9-engineered cell lines and animal models provides definitive specificity controls

  • Multiple antibody validation strategies now include immunoprecipitation-mass spectrometry to confirm target binding and identify potential cross-reactive proteins

  • Standardized validation initiatives are promoting more rigorous antibody testing across the research community

• Improved Antibody Engineering:

  • Recombinant antibody technology has enabled production of more consistent and specific AKAP7 antibodies

  • Single-chain variable fragments (scFvs) and nanobodies against AKAP7 allow for better penetration in tissue samples and reduced background

  • Site-specific conjugation methods provide better-controlled antibody labeling for imaging and other applications

• Advanced Imaging Applications:

  • Proximity ligation assays using AKAP7 antibodies enable visualization of protein-protein interactions in situ with high specificity

  • Expansion microscopy combined with AKAP7 immunofluorescence provides super-resolution imaging of AKAP7 complexes

  • Live-cell imaging using cell-permeable nanobodies allows dynamic studies of AKAP7 in living systems

• Multiplexed Detection Systems:

  • Mass cytometry (CyTOF) using metal-tagged antibodies allows simultaneous detection of AKAP7 and dozens of other proteins

  • Multiplexed immunofluorescence using spectral unmixing enables co-detection of AKAP7 with multiple interaction partners

  • Sequential immunohistochemistry methods permit layered staining of tissue sections for AKAP7 and related proteins

• Spatial Omics Integration:

  • Spatial transcriptomics combined with AKAP7 immunofluorescence correlates protein localization with gene expression patterns

  • Digital spatial profiling allows region-specific, quantitative analysis of AKAP7 in heterogeneous tissue samples

  • In situ sequencing combined with protein detection provides multi-omic analysis at single-cell resolution

• Advanced Quantification Methods:

  • Digital image analysis with machine learning algorithms improves quantification of AKAP7 staining patterns

  • Automated western blot systems provide more reproducible quantification of AKAP7 protein levels

  • Quantitative mass spectrometry-based immunoprecipitation enables precise measurement of AKAP7-interacting proteins

These methodological advances are transforming how researchers can use AKAP7 antibodies to address increasingly sophisticated questions about compartmentalized signaling in complex biological systems.

How can researchers select the most appropriate AKAP7 antibody for their specific research question?

Selecting the most appropriate AKAP7 antibody requires careful consideration of several factors:

• Research Question Specificity:

  • For isoform-specific studies, choose antibodies raised against unique regions of particular isoforms

  • For total AKAP7 detection, select antibodies targeting common domains shared by all isoforms, such as the 12591-1-AP antibody that recognizes all AKAP7 isoforms

  • For phosphorylation studies, consider whether you need antibodies against AKAP7 itself or against its phosphorylated substrates

• Application Compatibility:

  • Verify validation data for your specific application (WB, IHC, IP, IF)

  • Review recommended dilutions for each application - for example, 1:500-1:1000 for WB, 1:20-1:200 for IHC, and 1:10-1:100 for IF/ICC with the 12591-1-AP antibody

  • Consider whether the antibody has been validated in published studies for your specific application

• Species Reactivity:

  • Confirm reactivity with your species of interest - the 12591-1-AP antibody shows reactivity with human, mouse, and rat samples

  • Be aware that expression patterns may differ between species - for example, AKAP7β shows predominant expression in human pancreas but has different patterns in rodents

• Epitope Information:

  • Review the immunogen sequence - for 12591-1-AP, this is amino acids 1-81 encoded by BC016927

  • Consider whether the epitope might be masked by protein interactions or post-translational modifications in your experimental context

  • Assess whether the epitope is conserved in your species of interest

• Validation Evidence:

  • Look for knockout validation data where the antibody has been tested in AKAP7 KO tissues

  • Check for published studies using the antibody - the 12591-1-AP antibody has been cited in multiple publications

  • Consider antibodies that have been validated by multiple methods (WB, IHC, IP, etc.)

• Technical Specifications:

  • Review antibody format (e.g., purified IgG, antigen affinity purified)

  • Check storage requirements and stability information - the 12591-1-AP antibody is stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 and is stable for one year at -20°C

  • Consider lot-to-lot consistency information from the manufacturer

By systematically evaluating these factors, researchers can select the AKAP7 antibody most likely to provide reliable results for their specific experimental context, tissue type, and research question.

What are the most critical controls and validation steps researchers should implement when publishing data generated using AKAP7 antibodies?

When publishing data generated using AKAP7 antibodies, researchers should implement these critical controls and validation steps:

• Knockout/Knockdown Controls:

  • Include AKAP7 knockout tissue/cells when available - these provide the most definitive control for antibody specificity

  • If knockout models are unavailable, RNA interference to reduce AKAP7 expression can serve as an alternative control

  • Show complete blots/images of both wild-type and knockout/knockdown samples to demonstrate specificity

• Multiple Antibody Validation:

  • Use at least two independent antibodies targeting different epitopes of AKAP7

  • Compare staining/detection patterns between antibodies to confirm consistency

  • If discrepancies exist, provide potential explanations and additional validation

• Correlation with mRNA Expression:

  • Include RT-PCR or RNA-seq data to correlate protein detection with mRNA expression

  • For isoform-specific studies, use quantitative RT-PCR with primers that distinguish between isoforms

  • Address any discrepancies between protein and mRNA data

• Positive and Negative Tissue Controls:

  • Include tissues known to express (brain, heart) or not express specific AKAP7 isoforms based on published data

  • Use these controls consistently across all experimental applications

• Antibody Protocol Details:

  • Provide complete methodological details including:

    • Antibody source, catalog number, and lot number

    • Dilutions used for each application

    • Incubation conditions (time, temperature, buffer composition)

    • Antigen retrieval methods (for IHC/IF)

    • Detection systems and imaging parameters

• Peptide Competition Assays:

  • When appropriate, include peptide competition experiments where the antibody is pre-incubated with the immunizing peptide

  • Show that the specific signal is eliminated or significantly reduced by peptide competition

• Recombinant Protein Standards:

  • For Western blotting, include recombinant AKAP7 protein standards when possible

  • Demonstrate that the antibody recognizes proteins of the expected molecular weight

• Immunoprecipitation Validation:

  • For interaction studies, verify that the antibody can specifically immunoprecipitate AKAP7

  • Confirm identity of immunoprecipitated proteins by mass spectrometry when possible

• Cross-Reactivity Assessment:

  • Test for potential cross-reactivity with other AKAP family members

  • Address any potential cross-reactivity issues in the discussion of results

• Reproducibility Information:

  • Include information about the reproducibility of key experiments

  • Provide sample sizes and statistical analyses

  • Address any observed variability in antibody performance