AKAP4 Antibody

What is AKAP4 and why is it significant in research?

AKAP4, also known as hAKAP82, is a major structural component of the sperm fibrous sheath that localizes to the entire length of the flagellum in human sperm. It belongs to the AKAP110 family and functions by binding to the regulatory subunit of protein kinase A (PKA), confining the holoenzyme to discrete cellular locations . While primarily studied in reproductive biology for its role in sperm motility, AKAP4 has gained significant attention as a potential cancer biomarker and therapeutic target in various malignancies . Its expression in cancer tissues despite being normally restricted to testicular germ cells makes it particularly valuable for targeted therapy research.

How many isoforms of AKAP4 exist and which ones are detected by commercial antibodies?

At least three isoforms of AKAP4 have been identified in humans. Most commercial antibodies, such as those from Boster Biological and Proteintech, are designed to detect the two longest isoforms . The calculated molecular weight of full-length AKAP4 is approximately 94-95 kDa, though the observed molecular weight in Western blot applications can appear at 82 kDa or 68 kDa depending on the specific isoform, post-translational modifications, and experimental conditions . When designing experiments, researchers should consider which isoforms are relevant to their research question and select antibodies accordingly.

What are the key differences between polyclonal and monoclonal AKAP4 antibodies for research applications?

Polyclonal AKAP4 antibodies, such as the rabbit polyclonal options available from Boster (A07362) and Proteintech (24986-1-AP), recognize multiple epitopes on the AKAP4 protein, potentially increasing sensitivity but with a higher risk of cross-reactivity . These are often generated using immunogens from the amino terminus of human AKAP4, with Boster's antibody specifically raised against a 17 amino acid peptide within the first 50 amino acids of AKAP4 . In contrast, monoclonal antibodies target a single epitope, offering greater specificity but potentially reduced sensitivity if that epitope is masked or altered. For novel applications or when working with complex samples, validation with both types may be advisable to confirm findings.

What are the validated applications for AKAP4 antibodies and their recommended dilutions?

AKAP4 antibodies have been validated for multiple experimental applications. Based on manufacturer recommendations and published research, the following applications and dilutions are suggested:

Researchers should note that optimal dilutions may vary depending on sample type, experimental conditions, and specific antibody lot . It is advisable to titrate the antibody for each application and tissue type.

How should researchers design RT-PCR and qPCR experiments to analyze AKAP4 expression?

For AKAP4 mRNA expression analysis, researchers should first isolate high-quality total RNA using validated kits such as the RNeasy Mini kit (Qiagen). For RT-PCR, published primers targeting human AKAP4 include:

Forward primer: 5′-TGATACTACAATGATGTCTGATGAT-3′
Reverse primer: 5′-GGAACTAGCAGCATCCTTGTAATCTTTATC-3′

When performing qPCR, it is essential to:

• Include appropriate housekeeping genes (e.g., β-actin) as internal controls

• Validate primer efficiency and specificity via melt curve analysis

• Run reactions in triplicate to ensure statistical reliability

• Include negative controls (e.g., normal tissues where AKAP4 is not expected)

• Normalize AKAP4 expression against the endogenous control

The amplicons should be sequenced or sub-cloned into vectors like TOPO (Invitrogen) to confirm sequence identity, especially when working with novel tissue types or in disease states .

What is the optimal protocol for detecting cellular localization of AKAP4 using immunofluorescence?

To determine AKAP4 cellular localization using immunofluorescence, researchers should consider the following methodological approach:

• Fix cells appropriately (4% paraformaldehyde for 15-20 minutes is often suitable)

• Permeabilize cells with 0.1-0.2% Triton X-100 for intracellular detection

• Block with 5% normal serum corresponding to the secondary antibody host

• Incubate with AKAP4 primary antibody at optimized dilution (typically overnight at 4°C)

• Wash thoroughly and incubate with fluorophore-conjugated secondary antibody (e.g., FITC-conjugated anti-rabbit IgG)

• Counterstain subcellular compartments as needed (e.g., calnexin for endoplasmic reticulum, GM130 for Golgi, lamin A/C for nuclear envelope)

• Mount with anti-fade medium containing DAPI for nuclear visualization

For surface localization studies, omit the permeabilization step and use flow cytometry as a complementary approach to quantify surface expression levels .

How can AKAP4 knockdown experiments be designed to study its role in cancer cell properties?

To investigate AKAP4's functional role in cancer, shRNA-mediated gene silencing approaches have proven effective. A comprehensive knockdown experiment should include:

• Design of multiple shRNA constructs targeting different regions of AKAP4 mRNA

• Transfection into cancer cell lines of interest (e.g., COLO 205 and HCT 116 for colorectal cancer studies)

• Verification of knockdown efficiency at both mRNA level (qRT-PCR) and protein level (Western blot)

• Assessment of phenotypic changes through multiple functional assays:

  • Cell viability assays (e.g., MTT assay)

  • Proliferation assays (cell counting at 24h, 48h, and 72h post-transfection)

  • Colony formation assays (at varying cell densities: 400, 800, and 1200 cells)

  • Migration and invasion assays

• Include appropriate controls (non-targeting shRNA) and perform experiments in triplicate

This systematic approach allows for comprehensive evaluation of AKAP4's contribution to malignant properties and potential as a therapeutic target.

What considerations are important when using AKAP4 antibodies to study its expression in cancer tissues versus normal tissues?

When investigating AKAP4 expression patterns across cancer and normal tissues, researchers should address several critical factors:

• Antibody validation: Confirm specificity using positive controls (testis tissue) and negative controls (normal tissues where AKAP4 is not expected)

• Sample processing: Standardize fixation and antigen retrieval methods, as AKAP4 detection can be influenced by these parameters (e.g., Proteintech recommends TE buffer pH 9.0 for antigen retrieval)

• Expression quantification: Use digital image analysis for IHC to generate reproducible H-scores or percentage positivity metrics

• Context interpretation: AKAP4 is normally expressed only in testicular germ cells, so its presence in other tissues may indicate ectopic expression

• Correlation with clinical parameters: Analyze AKAP4 expression in relation to tumor stage, grade, and patient outcomes

When publishing findings, researchers should report detailed methodological parameters including antibody catalog numbers, dilutions, incubation conditions, and scoring criteria to ensure reproducibility .

How can AKAP4 surface localization be effectively measured in cancer cell lines?

Surface localization of AKAP4 in cancer cells can be methodically assessed through flow cytometry using the following protocol:

• Harvest cells using non-enzymatic cell dissociation solution to preserve surface proteins

• Incubate live cells with anti-AKAP4 antibody (optimal concentration determined by titration)

• Avoid fixation/permeabilization to ensure only surface proteins are detected

• Use appropriate isotype control antibodies to establish background staining levels

• Apply fluorophore-conjugated secondary antibody (e.g., FITC-conjugated anti-mouse IgG)

• Analyze using flow cytometry with appropriate compensation controls

• Compare mean fluorescence intensity across different cell lines or treatment conditions

This approach has been successfully employed to demonstrate AKAP4 surface expression in colorectal cancer cell lines, making it a valuable technique for identifying potential targets for antibody-based therapeutics or diagnostics .

What are common causes of inconsistent Western blot results with AKAP4 antibodies and how can they be resolved?

Researchers frequently encounter discrepancies in AKAP4 detection by Western blot, particularly regarding molecular weight (ranging from 68-94 kDa in published reports). These inconsistencies may be addressed through:

• Sample preparation optimization:

  • Include protease inhibitors to prevent degradation

  • Use fresh samples whenever possible

  • Optimize protein extraction buffer for membrane proteins

• Electrophoresis and transfer conditions:

  • For this high molecular weight protein, use lower percentage SDS-PAGE gels (8-10%)

  • Extend transfer time or use specialized transfer systems for large proteins

  • Consider wet transfer rather than semi-dry for more complete transfer

• Antibody selection and dilution:

  • Test multiple antibodies targeting different epitopes

  • Optimize primary antibody concentration through titration experiments

  • Extend incubation time (overnight at 4°C may improve signal)

• Detection system sensitivity:

  • For low expression samples, use enhanced chemiluminescence substrates

  • Consider signal amplification methods for extremely low abundance

The discrepancy between calculated (94 kDa) and observed (68-82 kDa) molecular weights may reflect post-translational modifications, proteolytic processing, or isoform variation .

How can researchers validate the specificity of their AKAP4 antibody results?

To ensure experimental rigor when working with AKAP4 antibodies, multiple validation approaches should be employed:

• Positive and negative controls:

  • Use human testis tissue as a positive control

  • Include normal colon epithelial cells or other AKAP4-negative tissues as negative controls

• Validation across techniques:

  • Confirm protein expression using multiple techniques (WB, IHC, IF)

  • Correlate protein data with mRNA expression (RT-PCR or qPCR)

• Blocking peptide competition:

  • Pre-incubate antibody with blocking peptide (available for some commercial antibodies)

  • Observe loss of specific signal while non-specific binding remains

• Genetic approaches:

  • Use AKAP4 knockdown/knockout samples to confirm specificity

  • Overexpression systems to validate antibody detection threshold

• Alternative antibodies:

  • Compare results using antibodies targeting different epitopes

  • Test both monoclonal and polyclonal antibodies when possible

These complementary approaches help distinguish true AKAP4 signal from potential cross-reactivity or artifacts.

What optimization strategies are recommended for immunohistochemical detection of AKAP4 in formalin-fixed, paraffin-embedded (FFPE) tissues?

Optimizing AKAP4 immunohistochemistry in FFPE tissues requires methodical approach to several variables:

• Antigen retrieval optimization:

  • Compare heat-induced epitope retrieval methods (microwave, pressure cooker, water bath)

  • Test both citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0) as recommended by Proteintech

  • Optimize retrieval time (typically 10-30 minutes)

• Antibody concentration and incubation:

  • Start with manufacturer-recommended dilution range (1:20-1:200 for Proteintech antibody)

  • Test both room temperature (1-2 hours) and 4°C overnight incubation

  • Consider signal amplification systems for weak expression

• Detection system selection:

  • Compare polymer-based detection systems with avidin-biotin methods

  • For dual staining, select enzyme combinations with differential substrates

• Counterstaining optimization:

  • Adjust hematoxylin timing to prevent obscuring weak positive signals

  • Consider alternative counterstains for special applications

• Control implementation:

  • Include on-slide positive control (testis tissue)

  • Run antibody diluent-only negative controls

  • Consider absorption controls with immunizing peptide when available

Thorough documentation of optimized conditions is essential for reproducibility across experiments and research groups.

How might AKAP4 antibodies be utilized in developing cancer immunotherapeutics or diagnostics?

AKAP4's unique expression pattern—absent in most normal tissues but present in various cancers—positions it as an attractive target for immunotherapeutic approaches. Researchers could explore:

• CAR-T cell development:

  • Use AKAP4 antibodies to isolate scFv sequences for chimeric antigen receptor construction

  • Test efficacy in preclinical models against AKAP4-expressing tumors

• Antibody-drug conjugates (ADCs):

  • Conjugate cytotoxic payloads to AKAP4-targeting antibodies

  • Assess internalization efficiency using fluorescently-labeled antibodies

• Diagnostic applications:

  • Develop immunoassays for detecting AKAP4 in circulating tumor cells

  • Create antibody-based imaging agents for tumor visualization

• Biomarker validation:

  • Standardize AKAP4 detection in tissue microarrays across multiple cancer types

  • Correlate expression with treatment response and survival outcomes

This research direction would require validation of antibody specificity across diverse cancer samples and careful assessment of any low-level expression in normal tissues to avoid off-target effects .

What are the considerations for analyzing post-translational modifications of AKAP4 with antibody-based methods?

Post-translational modifications (PTMs) of AKAP4 may significantly impact its function and localization in both normal and pathological contexts. Researchers investigating PTMs should consider:

• Specific PTM antibodies:

  • Select antibodies that recognize AKAP4 with specific modifications (phosphorylation, glycosylation, etc.)

  • Validate using appropriate controls (phosphatase-treated samples for phospho-specific antibodies)

• Enrichment strategies:

  • Implement immunoprecipitation with AKAP4 antibodies followed by PTM-specific detection

  • Consider phospho-protein or glycoprotein enrichment prior to AKAP4 detection

• Mass spectrometry integration:

  • Use antibody-based purification followed by mass spectrometry to identify novel PTMs

  • Compare modification patterns between normal and pathological samples

• Functional correlation:

  • Design experiments to determine how specific PTMs affect AKAP4's interaction with binding partners

  • Use site-directed mutagenesis to confirm the impact of specific modification sites

While commercial antibodies specifically targeting modified AKAP4 are currently limited, this represents an area for future reagent development to advance understanding of AKAP4 regulation .