AKAP13 Antibody

What applications are AKAP13 antibodies validated for?

AKAP13 antibodies have been validated for multiple applications across different experimental contexts:

When selecting an AKAP13 antibody, consider that different clones and formats have been validated for specific applications. For example, the rabbit polyclonal antibody (27947-1-AP) has been validated for detecting AKAP13 in HeLa cells via Western blot and in human breast, colon, and lung cancer tissues via immunohistochemistry . Similarly, the mouse monoclonal antibody (clone 5B7) has been confirmed for ELISA, immunofluorescence, and Western blot applications with human samples .

For optimal results, always verify the validation status of your specific antibody lot for your intended application and experimental system.

What are the recommended dilution ranges and protocols for AKAP13 antibodies?

Optimal dilution ranges vary by application and specific antibody preparation:

For Western blot protocols using rabbit polyclonal antibodies (27947-1-AP), antigen retrieval with TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may be used as an alternative . When performing immunohistochemistry, normal breast tissue serves as a positive control for AKAP13 detection, as described in methodologies for paraffin-embedded tissues .

For paraffin-embedded tissue sections, a validated protocol includes:

• Deparaffinization in xylene

• Rehydration through graded ethanol series

• Antigen retrieval using sodium citrate buffer (pH 6.0) at 95°C for 45 minutes

• Blocking in 1X Tris-buffered saline with 3% BSA/0.1% Tween-20

How should immunohistochemical detection of AKAP13 be optimized?

Optimizing AKAP13 detection by immunohistochemistry requires attention to several critical parameters:

• Antigen retrieval: For paraffin-embedded sections, sodium citrate buffer (pH 6.0) heated to 95°C for 45 minutes in a vegetable steamer has been validated. Some antibodies perform better with TE buffer (pH 9.0) .

• Blocking conditions: Use 1X Tris-buffered saline containing 3% BSA/0.1% Tween-20 with appropriate serum for 30 minutes .

• Antibody concentration: For rabbit polyclonal antibodies, a 1:50-1:500 dilution range is recommended, with specific validation required for each application .

• Controls: Include positive controls (normal breast tissue has been validated) and negative controls (secondary antibody only, without primary antibody, or preimmune sera) .

• Visualization: After primary and biotinylated secondary antibody incubation, standard streptavidin-biotin detection systems can be employed .

For detecting AKAP13 in fibroid and myometrial tissues, a peptide corresponding to AKAP13 protein (CREKEKDKIKEKEKDSKEKEKDKKTLNGHTF) was successfully used to generate polyclonal antiserum (6969) with binding confirmed via ELISA .

What epitopes and immunogens are used for generating AKAP13 antibodies?

Different manufacturers target various epitopes within the AKAP13 protein:

The specific epitope selection has important implications for which domains and functions of AKAP13 the antibody can detect. For instance, antibodies targeting the carboxyl domain may be particularly useful for studying interactions with steroid hormone receptors, as this region contains a nuclear receptor interaction domain (NRID) that binds multiple steroid hormone receptors .

The mouse monoclonal antibody (clone 5B7) targets the N-terminal region (amino acids 1-110) of AKAP13, with an immunogen sequence of "MKLNPQQAPLYGDCVVTVLLAEEDKAEDDVVFYLVFLGSTLRHCTSTRKVSSDTLETIAPGHDCCETVKVQLCASKEGLPVFVVAEEDFHFVQDEAYDAAQFLATSAGNQ" .

How can AKAP13 antibodies be used to investigate protein-protein interactions?

AKAP13 functions as a scaffold protein interacting with multiple partners. Investigating these interactions requires specific methodological approaches:

• Co-immunoprecipitation studies: AKAP13 antibodies have been successfully used to pull down AKAP13 and identify associated proteins. Research has demonstrated that AKAP13 interacts with:

  • Protein Kinase A (PKA) regulatory subunits

  • Estrogen receptor α (ERα)

  • mTORC1 complex components

  • Progesterone receptor B (PR-B)

• Glutathione S-transferase (GST)-binding assays: These have revealed that AKAP13 binds to PR-B through its carboxyl terminus, which contains a nuclear receptor interaction domain (NRID) .

• Investigation of phosphorylation states: When studying AKAP13's role in signaling, researchers have used HA-tagged Raptor co-expressed with Flag-tagged AKAP13, followed by immunoprecipitation and analysis using phospho-PKA substrate antibodies to detect Raptor Ser 791 phosphorylation .

For example, to investigate AKAP13's role in tamoxifen resistance, researchers demonstrated that "AKAP13 was found to interact with ERα as well as with a regulatory subunit of PKA. Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance" .

What are the considerations for detecting specific AKAP13 isoforms?

AKAP13 exists in multiple isoforms arising from alternative splicing, presenting challenges for specific detection:

• Antibody epitope selection: Choose antibodies targeting regions specific to your isoform of interest or common to all isoforms depending on your research question.

• Molecular weight verification: The calculated molecular weight of full-length AKAP13 is approximately 307-308 kDa, but shorter isoforms exist. Verify the expected molecular weight of your target isoform .

• Controls for specificity: When investigating specific isoforms, include positive controls of known isoform expression and validate with complementary techniques such as RT-PCR.

• Domain-specific detection: AKAP13 contains several functional domains including:

  • Rho-GEF domain

  • Nuclear receptor interaction domain (NRID) at the C-terminus

  • PKA-anchoring domain

For domain-specific studies, researchers have designed truncated versions of AKAP13 based on functional domains. For example, when mapping mTORC1 interaction sites on AKAP13, four different AKAP13 truncations were designed and analyzed .

How does AKAP13 expression vary across tissue types and disease states?

AKAP13 expression patterns show significant tissue and disease-specific variation:

• Cancer tissues: AKAP13 antibodies have demonstrated positive immunohistochemical staining in:

  • Breast cancer tissue

  • Colon cancer tissue

  • Lung cancer tissue

• Prognostic significance: In lung adenocarcinoma (LUAD), higher AKAP13 mRNA expression positively correlates with improved clinical survival rates. Four different LUAD cell lines (DFCI032, H2126, H2887, and A549) showed varying levels of AKAP13 expression, with H2887 and A549 having higher expression and lower mTORC1 activity .

• Uterine leiomyomata (fibroids): AKAP13 is overexpressed in fibroids and contributes to altered mechanotransduction. Studies in patients treated with ulipristal acetate (a selective progesterone receptor modulator) have examined AKAP13 expression using immunohistochemistry .

• Cardiac tissue: AKAP13-null mice showed deficient sarcomere formation and thin-walled developing hearts, leading to embryonic lethality at days 10.5-11.0, indicating an essential role in cardiac development .

Research has demonstrated that AKAP13 expression levels correlate with disease outcomes. For example, "Time to tumor progression (TTP) was estimated according to the Kaplan–Meier method for AKAP13 expression, segmenting the continuous variable in two groups (low vs. high)" .

What are the best storage and handling practices for AKAP13 antibodies?

Proper storage and handling of AKAP13 antibodies is critical for maintaining reactivity and specificity:

Most manufacturers recommend:

• Storing antibodies at -20°C for long-term storage

• Aliquoting to prevent repeated freeze-thaw cycles which can degrade antibody quality

• For working solutions, stability at 4°C is typically limited to 1-2 weeks

For the mouse monoclonal antibody (5B7), the recommendation is to "Aliquot and store at -20°C or -80°C. Avoid freeze-thaw cycles" . Similarly, for the rabbit polyclonal antibody (27947-1-AP), the storage guideline is "Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20°C storage" .

How can AKAP13 antibodies be used to investigate its role in progesterone receptor signaling?

AKAP13 has been identified as an important mediator of progesterone receptor (PR) signaling, particularly in uterine tissue. To investigate this role:

• Isoform-specific detection: Use antibodies that distinguish between PR-A and PR-B (such as Cell Signaling Technology clone 6A1 for both isoforms, and clone C1A2 for PR-B specific detection) .

• Co-immunoprecipitation: AKAP13 antibodies can be used to pull down the protein complex and examine its association with PR. Glutathione S-transferase (GST)-binding assays have revealed that "AKAP13 was bound to PR-B through its carboxyl terminus" .

• Luciferase reporter assays: Research has shown that "AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A" . This can be used to functionally validate antibody-based findings.

• Phosphorylation studies: When investigating signaling pathways, researchers found that "Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect" .

• Clinical sample analysis: In studies of patients treated with ulipristal acetate (a selective PR modulator), AKAP13 expression was examined in fibroid samples using immunohistochemistry, providing insights into therapeutic mechanisms .

The carboxyl domain of AKAP13 contains a nuclear receptor interaction domain (NRID) that allows it to bind multiple steroid hormone receptors including PR-B, enabling it to "enhance ligand-dependent transcriptional activation by several nuclear hormone receptors" .

What methodological approaches can resolve contradictory data on AKAP13 expression in patient samples?

Resolving contradictory AKAP13 expression data requires rigorous methodological approaches:

• Multi-antibody validation: Use antibodies targeting different epitopes of AKAP13 to confirm expression patterns. Compare results between polyclonal antibodies (which recognize multiple epitopes) and monoclonal antibodies (which target a single epitope).

• Complementary techniques: Correlate protein detection (via antibodies) with mRNA analysis. For example, researchers analyzing AKAP13 in lung cancer cell lines validated their findings by showing that "RNA sequencing data, immunoblot, and mRNA analysis corresponded with DFCI032 and H2126 having low AKAP13 expression, whereas H2887 and A549 have high AKAP13 expression" .

• Functional validation: Correlate expression with functional readouts. For instance, "cells with higher AKAP13 expression showed lower mTORC1 activity" and "lung cancer cells (H2887, A549) with high AKAP13 expression formed fewer colonies than lung cancer cells (DFC1032, H2126) with low AKAP13 expression" .

• Patient stratification: Carefully categorize patient samples. For example, "Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13" .

• Correlation with clinical outcomes: Link expression patterns to clinical data: "AKAP13 mRNA expression levels correlate with poor outcome in patients who received tamoxifen treatment in the metastatic setting" .

• Specific isoform detection: Distinguish between AKAP13 variants, as alternative splicing can produce different isoforms with potentially different functions or expression patterns .

How can AKAP13 antibodies be employed in investigating tamoxifen resistance mechanisms in breast cancer?

AKAP13 has emerged as a critical mediator of tamoxifen resistance in breast cancer through its interaction with estrogen receptor α (ERα). Key methodological approaches include:

• Phosphorylation status analysis: AKAP13 antibodies can be used alongside phospho-specific antibodies for ERα. Research has shown that "AKAP13 mRNA levels correlate with ERα Serine 305 phosphorylation in breast tumor samples, suggesting a functional connection between these two events" .

• Protein interaction studies: Co-immunoprecipitation experiments have demonstrated that "AKAP13 was found to interact with ERα as well as with a regulatory subunit of PKA" .

• Knockdown experiments: Functional validation through siRNA approaches revealed that "Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process" .

• Clinical correlation studies: AKAP13 expression can be correlated with treatment outcomes: "Time to tumor progression (TTP) was estimated according to the Kaplan–Meier method for AKAP13 expression, segmenting the continuous variable in two groups (low vs. high)" .

• Mechanistic pathway analysis: Pathway analysis combining antibody-based methods with functional assays showed that "the PKA-anchoring protein AKAP13 is essential for the phosphorylation of ERαS305, which leads to tamoxifen resistance both in cell lines and tamoxifen-treated breast cancer patients" .

This research area exemplifies how AKAP13 antibodies can be integrated into translational research with potential therapeutic implications for hormone-resistant breast cancer.

What are the technical considerations for using AKAP13 antibodies in studying mTORC1 inhibition?

Investigating AKAP13's role in mTORC1 inhibition requires specialized technical approaches:

• Domain mapping: To identify interaction regions, researchers designed "four different AKAP13 truncations based on a previous study" to map where mTORC1 interacts with AKAP13 .

• Phosphorylation analysis: For studying PKA-mediated phosphorylation events, specialized approaches include:

  • Immunoprecipitation of HA-tagged Raptor co-expressed with Flag-tagged AKAP13

  • Analysis using phospho-PKA substrate antibodies (pPKA Sub (R-R-X-S*/T*)) that specifically recognize Raptor Ser 791 phosphorylation

• Genetic modification systems: CRISPR-based approaches have been used to generate homozygous Raptor S791A knock-ins (clones S791A-1 and S791A-2) to confirm that "Flag-tagged AKAP13 overexpression decreased mTORC1 activity in wild type HEK293A cells, but not in HEK293A S791A-1 and S791A-2 cells" .

• Functional readouts: Several mTORC1-dependent processes can be measured:

  • Cell proliferation (significantly increased in AKAP13 KO cells)

  • Cell size (increased in AKAP13 KO cells)

  • Colony formation (increased in AKAP13 KO cells)

• Signal pathway integration: AKAP13 connects G-protein coupled receptor (GPCR) signaling to mTORC1 inhibition, where "AKAP13 acts as a scaffold for PKA and mTORC1, which results in Raptor Ser 791 phosphorylation and mTORC1 inhibition" .

What are the emerging applications of AKAP13 antibodies in cardiovascular research?

AKAP13 plays critical roles in cardiac development and function, with several emerging research applications:

• Developmental studies: AKAP13-null mice exhibited "deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5–11.0," indicating essential roles in cardiomyocyte development .

• Transcriptional regulation: AKAP13 antibodies can be used to investigate its upstream regulation of critical cardiac transcription factors: "Disruption of Akap13 was accompanied by reduced expression of Mef2C" and "overexpression of AKAP13 augmented MEF2C-dependent reporter activity" .

• G-protein coupled signaling: AKAP13 coordinates "Gα12 and Rho signaling to an essential transcription program in developing cardiomyocytes" .

• Cytoskeletal organization: Given AKAP13's role as a Rho Guanine Nucleotide Exchange Factor, antibodies can be used to study how it regulates actin dynamics in cardiac cells. This is particularly relevant as "an essential function of AKAP13 in mechanotransduction and cell survival has been described in human stem cells in culture" .

• Mechanical stress responses: AKAP13 has been implicated in cellular responses to mechanical cues, with research showing that "uterine fibroids exist in an environment of increased mechanical stress but have a decreased response to mechanical cues" .