AKAP14 Antibody

Basic Research Questions

• What is AKAP14 and what is its biological function?

  AKAP14 (A-kinase anchor protein 14) is a member of the AKAP family that binds to type II regulatory subunits of protein kinase A (PKA) and confines the holoenzyme to discrete locations within the cell. The protein anchors PKA in ciliary axonemes and may play a role in regulating ciliary beat frequency .

  In humans, the canonical AKAP14 protein has a reported length of 197 amino acid residues and a molecular mass of approximately 23 kDa . It is primarily localized in the cytoplasm and is notably expressed in the testis, nasopharynx, fallopian tube, and bronchus . Up to three different isoforms have been reported for this protein, with alternate transcriptional splice variants characterized .

• What applications are AKAP14 antibodies validated for?

  AKAP14 antibodies have been validated for multiple applications, with Western Blot (WB) being the most common. Based on the available research data, validated applications include:

  Application	Commonly Used Dilutions
  Western Blot (WB)	1:500-1:5,000
  Immunohistochemistry (IHC)	1:20-1:500
  Immunocytochemistry (ICC)	1:50-1:200
  Immunofluorescence (IF)	1:200-1:1,000
  ELISA	1:5,000
  Immunoprecipitation (IP)	1:10

  The optimal dilution should be determined empirically for each specific antibody and experimental condition .

• What tissues and cell lines express AKAP14?

  AKAP14 shows a tissue-specific expression pattern:

  High expression:

  • Testis

  • Fallopian tube

  • Nasopharynx

  • Bronchus

  Cell lines with confirmed expression:

  • Jurkat cells

  • HUVEC cells

  • NCI-H460 cells

  • A549 cells

  Immunohistochemistry analysis has also detected AKAP14 in human kidney, brain, lung, ovary, placenta, and prostate tissues, with varying expression levels .

Advanced Research Questions

• How should researchers validate the specificity of AKAP14 antibodies?

  For rigorous AKAP14 antibody validation, implement the following methodology:

  1. Positive and negative controls: Use tissues with known high expression (testis, fallopian tube) and low expression (prostate) of AKAP14 .

  2. Orthogonal validation: Compare antibody detection with mRNA expression data. RNA-seq data for AKAP14 should correlate with protein detection patterns .

  3. Western blot validation: Confirm specificity by detecting a band at the predicted molecular weight (23 kDa), though the observed weight may be ~28 kDa . Lysates from Jurkat cells, human testis tissue, or human fetal kidney tissue serve as reliable positive controls .

  4. Knockdown/knockout validation: When possible, use siRNA knockdown or CRISPR knockout of AKAP14 in relevant cell lines to confirm specificity.

  5. Multiple antibody validation: Compare results using antibodies targeting different epitopes of AKAP14 (N-terminal vs. C-terminal) .

• How do monoclonal and polyclonal AKAP14 antibodies compare in different experimental contexts?

  Monoclonal and polyclonal AKAP14 antibodies have distinct characteristics affecting their performance:

  Monoclonal antibodies (e.g., [EPR12955], [ARC2344]):

  • Provide higher specificity with less background

  • Show improved lot-to-lot consistency

  • Typically validated for fewer applications (mainly WB, ICC/IF, IP)

  • Often target specific epitopes (like C-terminal regions)

  • Suitable for quantitative analyses requiring high reproducibility

  Polyclonal antibodies:

  • Recognize multiple epitopes, potentially enhancing signal in low-expression contexts

  • Validated for more diverse applications (WB, IHC, ICC/IF, ELISA)

  • Show broader reactivity across species (human, mouse, rat)

  • May have batch-to-batch variability

  • Better for detecting native proteins or denatured isoforms

  Selection should be based on experimental requirements, with monoclonals preferred for quantitative work and polyclonals for detection of low-abundance targets or cross-species research.

• What are the optimal antigen retrieval methods for AKAP14 detection in FFPE tissues?

  For effective AKAP14 detection in formalin-fixed paraffin-embedded (FFPE) tissues:

  Recommended methods:

  • Heat-Induced Epitope Retrieval (HIER) with TE buffer at pH 9.0 is the primary recommended method .

  • Alternative approach: Citrate buffer at pH 6.0 may also be effective .

  Protocol considerations:

  • Optimal antibody dilution range for IHC: 1:20-1:200

  • Perform antigen retrieval prior to antibody incubation

  • Use appropriate positive control tissues (fallopian tube shows high expression, prostate shows low expression)

  Validation approach:

  • Compare IHC results with orthogonal data like RNA-seq expression patterns

  • Use multiple antibodies targeting different epitopes when possible

  • Include both positive and negative control tissues

• How can researchers detect different AKAP14 isoforms?

  Detection of AKAP14 isoforms requires strategic planning:

  Isoform characteristics:

  • Up to three different isoforms have been reported for AKAP14

  • Alternate transcriptional splice variants encode different isoforms

  Methodological approaches:

  1. Epitope-specific antibodies: Use antibodies targeting different regions of AKAP14:

     • N-terminal antibodies (amino acids 1-50)

     • C-terminal antibodies

  2. Immunoblotting optimization:

     • Use gradient gels (10-20%) to better resolve proteins of similar molecular weights

     • Extended run times can help separate closely migrating isoforms

     • Compare apparent molecular weights with predicted sizes (canonical form: 23 kDa)

  3. RT-PCR complementation:

     • Design primers spanning different exon junctions to detect specific splice variants

     • Correlate protein detection with transcript expression

  4. Mass spectrometry validation:

     • For definitive isoform identification, immunoprecipitate AKAP14 and analyze by mass spectrometry

     • This can provide unambiguous identification of specific isoforms

• What is the significance of AKAP14 in ciliary function research?

  AKAP14's role in ciliary function presents important research implications:

  Functional significance:

  • AKAP14 anchors PKA in ciliary axonemes

  • It likely plays a regulatory role in ciliary beat frequency

  • Understanding this function is relevant to respiratory, reproductive, and developmental biology

  Research approaches:

  1. Localization studies:

     • Use anti-AKAP14 antibodies for immunofluorescence microscopy to visualize distribution in ciliated cells

     • Co-localization studies with ciliary markers can confirm axonemal localization

  2. Functional assays:

     • Measure ciliary beat frequency after AKAP14 knockdown/overexpression

     • Analyze PKA activity and localization in the presence/absence of AKAP14

  3. Interaction studies:

     • Use co-immunoprecipitation with anti-AKAP14 antibodies to identify interaction partners

     • Validate interactions with PKA regulatory subunits and other ciliary proteins

  4. Disease models:

     • Investigate AKAP14 expression and function in models of ciliopathies

     • Study potential roles in respiratory conditions affecting ciliary function

• How should researchers troubleshoot cross-reactivity issues with AKAP14 antibodies?

  When encountering cross-reactivity with AKAP14 antibodies, implement this systematic troubleshooting approach:

  Identify potential cross-reactivity:

  • Compare observed band patterns with predicted molecular weight (23 kDa)

  • Note that AKAP14 may appear at approximately 28 kDa in some systems

  • Unexpected bands may indicate cross-reactivity with related AKAP family proteins

  Optimization strategies:

  1. Antibody selection:

     • Switch to monoclonal antibodies for higher specificity

     • Choose antibodies validated through orthogonal methods

     • Consider antibodies targeting unique regions of AKAP14

  2. Protocol adjustments:

     • Increase blocking time/concentration (5% BSA or milk)

     • Optimize primary antibody dilution (test series from 1:500 to 1:5,000)

     • Add detergent (0.1-0.3% Triton X-100) to reduce non-specific binding

     • Reduce antibody incubation time or switch to 4°C overnight

  3. Validation experiments:

     • Include knockout/knockdown controls when possible

     • Pre-absorb antibody with immunizing peptide if available

     • Compare results across multiple anti-AKAP14 antibodies targeting different epitopes

     • Confirm specificity with immunoprecipitation followed by mass spectrometry

Technical Considerations

• What control samples are essential when working with AKAP14 antibodies?

  Proper experimental controls are critical for AKAP14 antibody validation:

  Positive controls:

  • Cell lines: Jurkat cells show reliable AKAP14 expression

  • Tissues: Human testis, fallopian tube, and bronchus display high expression

  • Lysates: Human fetal kidney tissue lysate

  Negative controls:

  • Primary antibody controls: Replace primary antibody with same-species IgG at equivalent concentration

  • Absorption controls: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Low-expression tissues: Prostate tissue shows minimal AKAP14 expression and can serve as a relative negative control

  Technical controls:

  • Loading controls: Use housekeeping proteins (β-actin, GAPDH) to normalize protein loading

  • Secondary antibody controls: Omit primary antibody to detect non-specific secondary binding

  • Isotype controls: Rabbit IgG isotype controls are available (A82272, A17360)

• How can researchers determine the optimal fixation methods for AKAP14 immunocytochemistry?

  Optimizing fixation for AKAP14 immunocytochemistry requires methodical testing:

  Recommended fixation approaches:

  1. Paraformaldehyde fixation:

     • Use 4% PFA for 10-15 minutes at room temperature

     • Suitable for most cell types including ciliated cells

  2. Methanol fixation:

     • 100% methanol at -20°C for 5-10 minutes

     • May better preserve some epitopes while potentially disrupting others

  3. Methanol-acetone mixture:

     • 1:1 methanol:acetone at -20°C for 5 minutes

     • Can improve antigen accessibility

  Optimization protocol:

  • Test multiple fixation methods side-by-side

  • Include permeabilization step (0.1-0.3% Triton X-100) for cytoplasmic targets

  • Try different antibody dilutions (1:50-1:200 for ICC)

  • Include both positive control cells (Jurkat) and negative controls

  Evaluation criteria:

  • Signal-to-noise ratio

  • Subcellular localization pattern (cytoplasmic for AKAP14)

  • Preservation of cellular morphology

  • Consistency with literature reports