AKAP8L Antibody

What is AKAP8L and why is it a significant research target?

AKAP8L (A-kinase anchoring protein 8-like) belongs to the AKAP family and plays critical roles in various cellular processes. It contains two C2H2 AKAP95-type zinc fingers and is primarily localized in the nuclear matrix. AKAP8L is involved in multiple biological functions including constitutive transport element (CTE)-mediated gene expression, nuclear envelope breakdown, chromatin condensation, and potentially regulating the initiation phase of DNA replication when associated with TMPO-beta . Recent studies have identified AKAP8L as a potential biomarker for cancer diagnosis and prognosis, particularly in renal clear cell carcinoma (KIRC) .

What applications are AKAP8L antibodies typically used for?

AKAP8L antibodies are primarily used in applications including:

• Western Blot (WB): For detecting denatured AKAP8L protein in cell lysates (typically using 1:200-1:1000 dilution)

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualizing cellular localization (typically using 1:20-1:200 dilution)

• Immunohistochemistry (IHC): For detection in paraffin sections or frozen tissue samples

• ELISA: For quantitative detection of antigenic peptide

What is the molecular weight of AKAP8L and how does this affect antibody selection?

AKAP8L has a calculated molecular weight of 72 kDa, which is also the observed molecular weight in most experimental settings . When selecting antibodies, researchers should confirm that their antibody of choice detects proteins at this molecular weight. Variation from the expected weight may indicate post-translational modifications, protein degradation, or non-specific binding, requiring further validation steps including knockout/knockdown controls.

How should AKAP8L antibodies be stored and handled for optimal performance?

Most commercial AKAP8L antibodies should be stored at -20°C and remain stable for approximately one year after shipment. The standard storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Aliquoting is generally unnecessary for -20°C storage. Some formulations may contain 0.1% BSA, particularly in smaller (20μl) sizes . Proper handling involves avoiding repeated freeze-thaw cycles and maintaining cold chain during experiments to preserve antibody functionality.

What are the optimal sample preparation methods for detecting AKAP8L in different cellular compartments?

For nuclear AKAP8L detection, careful nuclear extraction protocols are essential as AKAP8L primarily localizes to the nuclear matrix. The protocol should include:

• Cell lysis using an appropriate buffer (e.g., RIPA buffer with protease inhibitors)

• Nuclear isolation through differential centrifugation

• Nuclear membrane disruption with appropriate detergents

• Protein quantification to ensure equal loading

For immunohistochemistry applications with tissue samples:

• Fixation in 10% formalin for 24 hours

• Paraffin embedding

• Sectioning at approximately 3 μM thickness

• Antigen retrieval through autoclaving at 121°C for 10 minutes

• Blocking with Serum-Free Protein Block

• Pretreatment with 100% methanol containing 3% hydrogen peroxide

What controls should be included when using AKAP8L antibodies in research?

A comprehensive experimental design should include:

• Positive controls: Cell lines known to express AKAP8L, such as PC-3 cells for Western blot applications or HEK-293 cells for IF/ICC applications

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG)

  • AKAP8L knockdown/knockout samples when available

• Loading controls: For Western blot normalization (e.g., β-actin, GAPDH)

• Specificity controls: Pre-absorption with the immunizing peptide when available

How can researchers optimize AKAP8L antibody dilutions for different applications?

Optimization requires systematic titration experiments:

The optimal dilution provides maximum specific signal with minimal background. Researchers should note that optimal dilutions may vary depending on the sample type, fixation method, and detection system .

How can AKAP8L antibodies be used to investigate its role in cancer progression?

AKAP8L has demonstrated significant potential as a cancer biomarker. Research approaches include:

What techniques can be used to study AKAP8L protein-protein interactions?

Several methodologies can be employed:

• Co-immunoprecipitation (Co-IP): Demonstrated in studies showing AKAP8L interaction with mTORC1. For example, immunoprecipitation of HA-tagged Raptor has been shown to co-precipitate FLAG-tagged AKAP8L under normal cell culture conditions . This interaction increases under amino acid starvation conditions.

• Proximity Ligation Assay (PLA): For detecting in situ protein interactions with spatial resolution.

• Pull-down assays: Using recombinant AKAP8L domains to identify interaction partners.

• FRET/BRET analysis: For studying dynamic interactions in living cells.

• Mass spectrometry following immunoprecipitation: For unbiased identification of interaction partners.

When designing Co-IP experiments, researchers should consider both forward (IP of AKAP8L to detect binding partners) and reverse (IP of suspected binding partners to detect AKAP8L) approaches to confirm interactions .

How can researchers investigate the role of AKAP8L in R-loop regulation using antibodies?

Recent research has revealed AKAP8L's involvement in R-loop regulation:

• Chromatin Immunoprecipitation (ChIP): To identify AKAP8L binding sites on chromatin.

• DNA-RNA Immunoprecipitation (DRIP): Using S9.6 antibody (R-loop specific) in combination with AKAP8L antibodies to study their co-occurrence.

• Immunofluorescence co-localization: To visualize AKAP8L and R-loop structures or R-loop resolution proteins like DDX5.

• Functional studies: Using AKAP8L antibodies alongside knockdown experiments to correlate changes in R-loop formation with AKAP8L levels. Research has shown that AKAP8L knockdown perturbs genomic R-loop formation and gene transcription .

• Interaction studies: Investigating AKAP8L's association with R-loop resolution protein DDX5, which can be validated using co-immunoprecipitation approaches .

What are common issues when using AKAP8L antibodies and how can they be resolved?

When troubleshooting, researchers should systematically modify one variable at a time and document all changes to protocols.

How should researchers interpret AKAP8L expression in relation to immune cell infiltration?

AKAP8L expression has been correlated with immune cell infiltration, particularly in KIRC tumors. Analysis should consider:

• Positive correlations: AKAP8L expression has shown positive correlation with CD8 T cells, T helper cells, pDC cells, Tem cells, NK CD56bright cells, and NK cells .

• Negative correlations: AKAP8L expression negatively correlates with Eosinophils, T cells, Th1 cells, TFH cells, Mast cells, Neutrophils, DC cells, B cells, iDC cells, Th2 cells, Tgd cells, and Macrophages .

• Methodology: Single-sample gene set enrichment analysis (ssGSEA) can be used to analyze immune infiltration in relation to AKAP8L expression. Spearman correlation analysis should be employed to analyze correlations between AKAP8L and immune cell populations .

• Statistical evaluation: Differences between high and low AKAP8L expression groups should be analyzed using Wilcoxon test, with p<0.05 considered statistically significant .

These correlations suggest AKAP8L may influence cancer prognosis through modulation of the immune microenvironment.

How can researchers validate AKAP8L antibody specificity for their experimental system?

A comprehensive validation strategy includes:

• Western blot analysis: Confirming a single band at the expected molecular weight (72 kDa).

• Knockdown/knockout validation: Demonstrating reduced or absent signal following AKAP8L silencing or deletion.

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide should eliminate specific binding.

• Cross-reactivity testing: For antibodies predicting reactivity across species (such as pig, bovine, horse, sheep, rabbit, and dog ), researchers should validate reactivity in each species of interest.

• Multiple antibody validation: Using antibodies targeting different epitopes of AKAP8L to confirm observations.

• Immunoprecipitation-mass spectrometry: To confirm antibody pulls down the intended target.

How might AKAP8L antibodies be used in developing cancer diagnostics and prognostics?

AKAP8L shows significant potential as a biomarker:

• Diagnostic applications: With AUC values >0.9 for detecting testicular germ cell tumors (TGCT), liver hepatocellular carcinoma (LIHC), and rectum adenocarcinoma (READ), AKAP8L antibodies could be developed into diagnostic immunoassays .

• Prognostic stratification: Given its correlation with survival outcomes in KIRC, AKAP8L immunohistochemistry could help identify high-risk patients who might benefit from more aggressive treatment approaches .

• Therapeutic monitoring: Antibody-based detection of AKAP8L expression changes during treatment could serve as a pharmacodynamic marker.

• Multimarker panels: AKAP8L detection could be incorporated into multimarker panels to improve diagnostic accuracy.

• Liquid biopsy development: Research into detection of AKAP8L in circulating tumor cells or exosomes could enable less invasive diagnostics.

What emerging techniques might enhance AKAP8L research using antibodies?

Several cutting-edge approaches show promise:

• Single-cell proteomics: Examining AKAP8L expression at the single-cell level to understand cellular heterogeneity.

• Spatial transcriptomics with antibody validation: Correlating AKAP8L mRNA distribution with protein localization.

• Antibody-based CRISPR screening: Using AKAP8L antibodies to evaluate phenotypic changes following genetic perturbations.

• Intrabodies: Developing antibody fragments that can track AKAP8L in living cells.

• Antibody-drug conjugates: Exploring AKAP8L as a potential therapeutic target in cancers showing overexpression.

• Proximity-dependent biotinylation: Using antibodies to validate BioID or APEX2 studies of the AKAP8L interactome.

How can researchers integrate AKAP8L antibody-based findings with genomic and transcriptomic data?

Multi-omics integration strategies include:

• Correlation analysis: Between AKAP8L protein levels (detected by antibodies) and mRNA expression.

• Promoter methylation studies: AKAP8L promoter methylation can be analyzed using UALCAN (http://ualcan.path.uab.edu) and correlated with protein expression .

• Integration with differentially expressed genes (DEGs): Comparing high vs. low AKAP8L expression groups to identify co-regulated pathways.

• Co-expression network analysis: Identifying the top co-expressed genes with AKAP8L, including reported interactions with CLASRP, TAF1C, CLK3, ZNF276, and SNRNP70 .

• Functional enrichment analysis: GO and KEGG pathway analysis on DEGs between AKAP8L high and low expression groups has revealed associations with acute-phase response, cellular processes involved in reproduction, drug catabolism, and terpenoid metabolism .

This integrated approach provides a more comprehensive understanding of AKAP8L's biological significance and potential as a therapeutic target.