AKAP17A Antibody

What is AKAP17A and where is it localized in cells?

AKAP17A (also known as SFRS17A) is a protein encoded by the DXYS155E gene found in the pseudoautosomal region of the distal short arms of the X and Y chromosomes. It is ubiquitously expressed throughout the body . At the cellular level, AKAP17A is primarily localized in nuclear speckles . The protein functions as a scaffold, organizing protein complexes involved in signal transduction pathways, and is a key component of the spliceosome complex where it regulates alternative splicing in some mRNA precursors .

What are the key aliases and identifiers for AKAP17A?

AKAP17A is known by multiple aliases in scientific literature which can complicate literature searches. Common protein aliases include: 721P, A-kinase anchor protein 17A, AKAP-17A, B-lymphocyte antigen, Protein XE7, Protein kinase A-anchoring protein 17A, and Splicing factor, arginine/serine-rich 17A. Gene aliases include: CCDC133, CXYorf3, DXYS155E, PRKA17A, SFRS17A, and XE7Y. The UniProt ID for human AKAP17A is Q02040, and its Entrez Gene ID is 8227 .

What are the main applications of AKAP17A antibodies in research?

AKAP17A antibodies are valuable tools in multiple research applications. They can be used for Western blotting and immunofluorescence to study protein expression and localization . Additionally, they are effective in immunohistochemistry to examine tissue-specific expression patterns and in immunoprecipitation to isolate AKAP17A and its binding partners from cell lysates . Enzyme-linked immunosorbent assay (ELISA) is another common application, particularly useful for quantitative analyses .

What are the optimal conditions for AKAP17A antibody use in immunohistochemistry?

For immunohistochemistry applications, polyclonal AKAP17A antibodies are typically used at dilutions between 1:40 and 1:200 . Verified samples for AKAP17A antibody testing include human esophagus cancer tissue . When designing IHC experiments, it's important to consider tissue fixation methods, antigen retrieval techniques, and appropriate blocking solutions to minimize background staining. Researchers should include positive controls (tissues known to express AKAP17A) and negative controls (omitting primary antibody) in their experimental design.

What protocols are recommended for immunoprecipitation of AKAP17A?

For immunoprecipitation of AKAP17A, a validated protocol includes:

• Resuspend AKAP17A-transfected HEK293 EBNA cells in 10 volumes of 450 mM NaCl, 160 mM NaH₂PO₄ (pH 7.5)

• Sonicate the cells for 5 minutes on ice

• Centrifuge the lysate at 3,400 × g for 10 minutes followed by 12,000 × g for 10 minutes at 4°C

• Pre-clear the supernatant using protein G Dynabeads

• Couple the AKAP17A antibody to protein G Dynabeads using BS3 cross-linking reagent

• Incubate 1 mL of the pre-cleared lysate with 750 μg of antibody-coupled beads for 30 minutes

• Wash beads five times with PBS/0.05% Tween-20

• Elute antigens with reducing SDS/PAGE loading buffer at 95°C for 10 minutes

• Analyze by Western blotting

This method has been validated for both commercial anti-AKAP17A antibodies and recombinant antibody fragments.

What are the recommended dilutions for AKAP17A antibodies in various applications?

Optimal dilutions vary by application and specific antibody preparation:

These dilutions serve as starting points and should be optimized for specific experimental conditions.

How can researchers address cross-reactivity between AKAP17A and FAM84A antibodies?

Cross-reactivity between AKAP17A and FAM84A has been documented due to regions of high sequence homology. Specifically, the region between amino acids 310-318 in AKAP17A shares homology with amino acids 263-271 in FAM84A . This cross-reactivity can lead to false positive results if not properly controlled.

To address this issue:

• Perform sequence alignment analysis to identify potential cross-reactive epitopes

• Include FAM84A protein as a negative control when testing AKAP17A antibodies

• Consider using antibodies that target non-homologous regions of AKAP17A

• Validate antibody specificity using knockout/knockdown models

• Employ multiple antibodies targeting different epitopes to confirm findings

Mutation studies have shown that amino acid positions 263, 264, 268, and 271 in FAM84A are critical for antibody recognition, suggesting these positions should be avoided when designing epitope-specific antibodies .

What experimental approaches are recommended for studying AKAP17A's role in the spliceosome complex?

To investigate AKAP17A's function in the spliceosome:

• Co-immunoprecipitation studies: Use AKAP17A antibodies to pull down associated spliceosome components

• RNA immunoprecipitation: Identify RNA targets bound by AKAP17A

• Alternative splicing assays: Examine changes in splicing patterns following AKAP17A depletion or overexpression

• Confocal microscopy: Use immunofluorescence with AKAP17A antibodies to visualize co-localization with other splicing factors in nuclear speckles

• Protein-protein interaction mapping: Identify binding partners within the spliceosome complex

When designing these experiments, researchers should consider the dynamic nature of the spliceosome and possible cell type-specific functions of AKAP17A .

What is known about AKAP17A's involvement in disease states?

AKAP17A has been associated with several pathological conditions. Diseases linked to AKAP17A include Null-Cell Leukemia and Chronic Tic Disorder . Given its role in RNA processing and signaling pathways, investigating AKAP17A in disease models may provide valuable insights into pathological mechanisms.

When designing disease-related studies:

• Compare AKAP17A expression levels between normal and disease tissues

• Investigate alterations in AKAP17A's splicing regulatory function in disease states

• Examine potential mutations or polymorphisms in the AKAP17A gene

• Study the impact of disease-specific conditions on AKAP17A's protein interactions

• Consider AKAP17A as a potential biomarker for disease diagnosis or progression

How can researchers validate the specificity of AKAP17A antibodies?

Antibody validation is critical for ensuring experimental reproducibility. Methods to validate AKAP17A antibodies include:

• Western blot analysis: Confirm single band of expected molecular weight

• Immunoprecipitation followed by mass spectrometry: Verify that AKAP17A is captured

• Testing in AKAP17A knockout/knockdown models: Antibody signal should be reduced or absent

• Peptide competition assays: Pre-incubation with the immunizing peptide should block antibody binding

• Cross-reactivity testing: Check reactivity against related proteins like FAM84A

• Multiple antibody approach: Use antibodies from different sources targeting different epitopes

These validation steps are particularly important given the potential cross-reactivity issues with FAM84A.

What are common challenges when using AKAP17A antibodies and how can they be addressed?

Several challenges may arise when working with AKAP17A antibodies:

What storage conditions ensure optimal AKAP17A antibody performance?

Proper storage is essential for maintaining antibody functionality. Most AKAP17A antibodies should be stored at -20°C and are valid for 12 months when properly maintained . Avoid repeated freeze-thaw cycles, which can degrade antibody quality. Many commercially available AKAP17A antibodies are supplied in storage buffers containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (e.g., 50% glycerol) in phosphate-buffered solutions at pH 7.4 .

When working with the antibody, aliquot into smaller volumes to minimize freeze-thaw cycles, and keep on ice during experiments to prevent degradation.

How might AKAP17A antibodies be utilized in emerging research areas?

AKAP17A antibodies have potential applications in several emerging research fields:

• Single-cell analysis: Examining AKAP17A expression and localization in heterogeneous cell populations

• Proximity labeling approaches: Using AKAP17A antibodies in conjunction with BioID or APEX techniques to map protein interaction networks

• Chromatin immunoprecipitation sequencing (ChIP-seq): Investigating potential roles of AKAP17A in transcriptional regulation

• Liquid-liquid phase separation studies: Exploring AKAP17A's role in nuclear speckle formation and dynamics

• Therapeutic target validation: Assessing AKAP17A as a potential target in diseases where RNA processing is dysregulated

What are the latest methodological advances in AKAP17A research?

Recent methodological advances include:

• Development of conformation-specific antibodies that recognize distinct structural forms of AKAP17A

• Combined immunoprecipitation and mass spectrometry approaches to identify novel AKAP17A binding partners

• CRISPR-Cas9 generated cellular models for antibody validation and functional studies

• Super-resolution microscopy techniques to visualize AKAP17A within nuclear speckle subdomains

• Phospho-specific antibodies to detect post-translational modifications of AKAP17A

These advances provide researchers with powerful tools to investigate AKAP17A's complex roles in cellular processes and disease states.