Acetyl-RELA (K122) Antibody

What is the Acetyl-RELA (K122) Antibody and what specific modification does it detect?

The Acetyl-RELA (K122) Antibody is a polyclonal antibody derived from rabbit that specifically recognizes the acetylation of the lysine residue at position 122 in the RELA (p65) subunit of the NF-κB transcription factor complex. This antibody was developed using a synthesized peptide derived from the internal region of human NF-κB-p65 surrounding the K122 acetylation site . The specificity for this particular post-translational modification allows researchers to investigate acetylation-dependent regulation of NF-κB activity, which is crucial for understanding its role in various cellular processes including inflammation, immune response, and cell survival .

What is the significance of lysine 122 acetylation in RELA compared to other acetylation sites?

Lysine 122 represents one of several important acetylation sites on RELA that modulates distinct functions of NF-κB signaling. While acetylation at sites like K314/K315 influences late NF-κB-dependent gene expression as demonstrated in comparative studies , K122 acetylation has its own unique regulatory functions. Post-translational modifications of NF-κB, including acetylation at different lysine residues, have emerged as important regulatory mechanisms for determining the duration and strength of NF-κB nuclear activity as well as its transcriptional output . Each acetylation site appears to regulate different aspects of NF-κB function, including DNA binding capability, transcriptional activity, protein stability, and interactions with other regulatory proteins. This site-specific regulation creates a sophisticated control system for NF-κB-mediated gene expression programs.

What species reactivity has been validated for the Acetyl-RELA (K122) Antibody?

The Acetyl-RELA (K122) Antibody has been validated for reactivity with human, mouse, and rat samples . This cross-species reactivity makes it a versatile research tool for comparative studies across these mammalian models. The antibody's ability to recognize the acetylated K122 site across multiple species suggests a high degree of conservation in this regulatory mechanism throughout mammalian evolution, underscoring its biological importance. This multi-species applicability enables researchers to translate findings between different experimental models, which is particularly valuable for studies aiming to understand fundamental mechanisms of NF-κB regulation that are conserved across species.

What are the validated experimental applications for Acetyl-RELA (K122) Antibody?

The Acetyl-RELA (K122) Antibody has been validated for two primary applications: Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) . For ELISA applications, the recommended dilution is 1:10000, while for Western Blot applications, the recommended dilution range is 1:500-1:2000 . Western Blot analysis has been successfully performed on HepG2-UV cells and mouse brain cells, as demonstrated in the product documentation . These applications make the antibody suitable for both quantitative measurement of acetylated RELA levels and qualitative detection of this modified protein in complex biological samples. The antibody's performance in these applications provides researchers with reliable tools to investigate acetylation-dependent regulation of NF-κB activity in various experimental contexts.

How should researchers design experiments to detect RELA acetylation at K122 in cellular systems?

When designing experiments to detect RELA acetylation at K122, researchers should consider a multi-faceted approach:

• For overexpression studies, researchers can transfect cells (such as HEK293T) with expression vectors for T7-tagged RELA and p300 (an acetyltransferase known to acetylate NF-κB). This typically involves transfecting 1 μg of T7-tagged RELA and 4 μg of p300 expression vector DNA using calcium phosphate precipitation or other transfection methods .

• For endogenous RELA acetylation detection, researchers should consider stimulating cells with appropriate NF-κB activators (such as TNFα) to induce acetylation.

• Cell lysates should be prepared using buffers that preserve acetylation status, typically containing deacetylase inhibitors like trichostatin A (TSA) or nicotinamide.

• Western blot analysis can be performed using the Acetyl-RELA (K122) antibody at the recommended dilution of 1:500-1:2000 .

• For verification of specificity, researchers should include appropriate controls, such as samples treated with deacetylases or samples from cells expressing RELA with K122R mutation (which prevents acetylation at this site).

This methodological approach enables reliable detection and quantification of site-specific acetylation of RELA in various experimental conditions.

What is the recommended protocol for immunoprecipitation experiments using Acetyl-RELA (K122) Antibody?

While specific immunoprecipitation (IP) protocols for the Acetyl-RELA (K122) Antibody are not directly provided in the search results, researchers can adapt standard IP protocols based on general principles for acetylation-specific antibodies:

• Prepare cell lysates in a non-denaturing buffer containing protease inhibitors and deacetylase inhibitors (such as trichostatin A and nicotinamide) to preserve acetylation status.

• Pre-clear the lysate with Protein A/G beads to reduce non-specific binding.

• Incubate the pre-cleared lysate with Acetyl-RELA (K122) Antibody (typically 2-5 μg per 1 mg of protein) overnight at 4°C with gentle rotation.

• Add Protein A/G beads and incubate for an additional 2-4 hours.

• Wash the beads thoroughly with IP buffer to remove non-specific interactions.

• Elute bound proteins by boiling in SDS sample buffer and analyze by Western blot using a different RELA antibody to confirm specificity.

For researchers interested in detecting acetylation using non-radioactive methods, immunoprecipitation followed by Western blotting with anti-acetylated lysine antibodies provides a sensitive approach to monitor RELA acetylation status .

How does RELA K122 acetylation affect the expression profile of NF-κB target genes compared to other acetylation sites?

Research comparing different acetylation sites shows that each site has distinct effects on gene expression profiles. While K122 acetylation has its specific regulatory functions, studies on other acetylation sites like K314/K315 provide insights into how different modifications create unique gene expression signatures. For instance, site-specific mutation of p65 at lysines 314 and 315 enhances the expression of a subset of NF-κB target genes, including Mmp10 .

Analysis of gene expression profiles following TNFα treatment revealed significant differences between wild-type and mutant cell lines. The gene expression data shows that modifications at different lysine residues can lead to either upregulation or downregulation of specific gene sets. For example, after TNFα treatment, genes like Mmp13 (Matrix metallopeptidase 13) and Mmp10 (Matrix metallopeptidase 10) showed upregulation (2.222-fold and 1.829-fold change, respectively) in cells with mutations at specific lysine residues .

This differential gene regulation underscores the importance of site-specific acetylation in determining the functional outcome of NF-κB signaling, suggesting that K122 acetylation likely regulates its own specific subset of target genes.

How can researchers differentiate between the functional consequences of different RELA acetylation sites in complex biological systems?

To differentiate between the functional consequences of different RELA acetylation sites, researchers should employ several complementary approaches:

• Site-specific mutant expression: Generate RELA constructs with lysine-to-arginine mutations at specific sites (K122R, K310R, K314/315R, etc.) to prevent acetylation at those positions. Express these constructs in RELA-knockout or RELA-depleted cells to assess site-specific functions.

• ChIP-seq analysis: Perform chromatin immunoprecipitation followed by sequencing using site-specific acetylation antibodies to identify genomic binding sites that are specifically regulated by each acetylation event.

• RNA-seq comparative analysis: Compare gene expression profiles between cells expressing wild-type RELA and various acetylation-deficient mutants, as demonstrated in the research that identified differential gene expression patterns in KTR cell lines .

• Proteomic approaches: Use mass spectrometry-based techniques to identify protein interaction partners that specifically recognize RELA acetylated at different lysine residues.

• Functional assays: Develop specific reporter assays for genes known to be regulated by different acetylation events to directly measure the functional impact of each modification.

By combining these approaches, researchers can build a comprehensive understanding of how different acetylation events orchestrate distinct aspects of NF-κB-mediated cellular responses.

What is the relationship between RELA K122 acetylation and other post-translational modifications in the NF-κB signaling pathway?

The relationship between RELA K122 acetylation and other post-translational modifications (PTMs) represents a complex regulatory network within NF-κB signaling. While the provided search results don't specifically address the interplay between K122 acetylation and other PTMs, we can draw insights from the general principles of NF-κB regulation:

• Cross-talk with other acetylation sites: Acetylation at K122 likely functions in concert with other acetylation events on RELA, including those at K310, K314, and K315. These modifications may work synergistically or antagonistically to fine-tune NF-κB activity .

• Interactions with phosphorylation: NF-κB signaling is heavily regulated by phosphorylation events. Acetylation at K122 may influence or be influenced by phosphorylation of nearby residues, creating a complex code of modifications that dictate function.

• Impact on ubiquitination and protein stability: Acetylation can sometimes compete with ubiquitination for the same lysine residues, potentially affecting protein stability and turnover. K122 acetylation might protect RELA from degradation by preventing ubiquitination.

• Influence on methylation: As both acetylation and methylation can occur on lysine residues, there may be competition between these modifications at K122 or cooperation with methylation at other sites .

Understanding these complex interrelationships requires sophisticated experimental approaches combining site-specific antibodies, mass spectrometry, and functional assays to dissect the spatiotemporal dynamics of multiple PTMs in response to different stimuli.

What are the common technical challenges when using Acetyl-RELA (K122) Antibody in Western blot analysis, and how can they be overcome?

When working with Acetyl-RELA (K122) Antibody in Western blot applications, researchers may encounter several technical challenges:

• Low signal intensity: This can be addressed by:

  • Optimizing antibody concentration within the recommended range (1:500-1:2000)

  • Increasing protein loading amount

  • Extending primary antibody incubation time (overnight at 4°C)

  • Using enhanced chemiluminescence (ECL) detection reagents with higher sensitivity

  • Ensuring samples are properly prepared with deacetylase inhibitors to preserve acetylation status

• High background: To reduce non-specific binding:

  • Increase blocking time or concentration (5% BSA or milk is typically effective)

  • Add 0.05-0.1% Tween-20 in wash buffers

  • Optimize antibody dilution, starting at 1:2000 and adjusting as needed

  • Consider using more stringent washing conditions

• Cross-reactivity: To ensure specificity:

  • Include appropriate negative controls (e.g., RELA knockout cells or K122R mutant)

  • Pre-absorb the antibody with the non-acetylated peptide if available

  • Use validated positive controls like UV-treated HepG2 cells, which have been shown to work in product testing

• Inconsistent results: For better reproducibility:

  • Standardize lysate preparation methods, including consistent use of deacetylase inhibitors

  • Maintain consistent stimulation conditions when inducing acetylation

  • Use internal loading controls to normalize between samples

  • Consider using fresh antibody aliquots to avoid freeze-thaw cycles

By systematically addressing these potential issues, researchers can optimize their Western blot protocols for reliable detection of RELA K122 acetylation.

How can researchers validate the specificity of signals detected with the Acetyl-RELA (K122) Antibody?

Validating the specificity of signals detected with the Acetyl-RELA (K122) Antibody is crucial for ensuring reliable experimental results. Researchers should implement several validation strategies:

• Genetic validation:

  • Use RELA knockout or knockdown cells as negative controls

  • Express RELA with K122R mutation (preventing acetylation at this site) to confirm signal specificity

  • Perform rescue experiments with wild-type RELA in knockout cells to restore the signal

• Pharmacological validation:

  • Treat cells with histone deacetylase (HDAC) inhibitors to increase global acetylation levels

  • Use specific p300/CBP inhibitors to reduce RELA acetylation

  • Compare acetylation patterns in cells treated with or without NF-κB activators like TNFα

• Peptide competition assays:

  • Pre-incubate the antibody with excess acetylated K122 peptide before immunoblotting

  • The specific signal should be reduced or eliminated if the antibody is specific

  • Include a non-acetylated peptide control that should not compete for binding

• Cross-validation with mass spectrometry:

  • Immunoprecipitate RELA and analyze by mass spectrometry to confirm acetylation at K122

  • This approach can simultaneously identify other modifications that may be present

• Multi-antibody approach:

  • Compare results with a general anti-acetyl-lysine antibody after RELA immunoprecipitation

  • Use a different antibody specific for total RELA to confirm that the detected protein is indeed RELA

These validation approaches, particularly when used in combination, provide strong evidence for signal specificity and increase confidence in experimental findings related to RELA K122 acetylation.

What methodological adaptations are recommended when studying RELA K122 acetylation in different cell types or tissue samples?

When studying RELA K122 acetylation across different biological systems, several methodological adaptations are recommended:

• Cell type-specific optimizations:

  • Adjust cell lysis conditions based on cell type (e.g., more stringent lysis for tough tissues, gentler conditions for fragile cells)

  • Optimize NF-κB stimulation protocols based on cell-specific receptor expression and signaling kinetics

  • Consider using cell type-specific transfection methods for overexpression studies

• Tissue sample processing:

  • For tissue samples, implement rapid preservation techniques to maintain acetylation status

  • Include additional protease and deacetylase inhibitors in tissue homogenization buffers

  • Consider using specialized extraction buffers optimized for nuclear proteins from tissues

• Species-specific considerations:

  • While the antibody is reactive with human, mouse, and rat samples , subtle differences in protein sequences may affect antibody affinity

  • Validate the antibody in each species before conducting comparative studies

  • Consider using species-specific positive controls (e.g., UV-treated HepG2 cells for human, UV-treated mouse brain cells for mouse)

• Immunohistochemistry adaptations:

  • For tissue sections, optimize antigen retrieval methods to expose the K122 epitope

  • Test different fixation protocols to preserve acetylation while maintaining tissue architecture

  • Consider using tyramide signal amplification for detecting low-abundance acetylated proteins

• Protein extraction modifications:

  • For samples with high deacetylase activity, increase the concentration of deacetylase inhibitors

  • For adipose or lipid-rich tissues, include additional centrifugation steps to remove lipids

  • For muscle or fibrous tissues, consider using mechanical homogenization followed by sonication

These adaptations should be systematically tested and optimized for each experimental system to ensure reliable and reproducible detection of RELA K122 acetylation across diverse biological contexts.

How can Acetyl-RELA (K122) Antibody be integrated into multi-omics research approaches?

The Acetyl-RELA (K122) Antibody can serve as a valuable tool in multi-omics research strategies by providing site-specific information about this critical post-translational modification. Integration can be achieved through several approaches:

• ChIP-seq integration: Use the antibody for chromatin immunoprecipitation followed by sequencing to identify genomic regions specifically bound by K122-acetylated RELA. This data can be integrated with RNA-seq to correlate binding with gene expression changes, similar to approaches that have revealed differential gene regulation by various NF-κB modifications .

• Proteomics coupling: Combine immunoprecipitation using the Acetyl-RELA (K122) Antibody with mass spectrometry to identify proteins that specifically interact with RELA when acetylated at K122. This interactome can reveal new functional connections and regulatory mechanisms.

• Single-cell applications: Adapt the antibody for single-cell proteomics or CyTOF (Cytometry by Time-of-Flight) to examine cell-to-cell heterogeneity in RELA K122 acetylation status within complex tissues or cell populations.

• Temporal dynamics studies: Use the antibody in time-course experiments combined with phospho-proteomics to understand the sequence and interdependence of different post-translational modifications during NF-κB activation.

• Spatial omics integration: Combine immunofluorescence using the antibody with spatial transcriptomics to correlate RELA K122 acetylation status with local gene expression patterns in tissue sections.

These integrated approaches can provide comprehensive insights into how K122 acetylation coordinates with other cellular mechanisms to orchestrate complex biological responses, moving beyond single-modification studies toward understanding systems-level regulation.

What are the implications of RELA K122 acetylation in disease pathogenesis based on current research?

While the search results don't directly address disease associations with RELA K122 acetylation, we can infer potential implications based on the broader role of NF-κB in disease processes and the specific gene expression changes associated with NF-κB acetylation:

• Inflammatory diseases: Given that NF-κB is a master regulator of inflammation, dysregulation of K122 acetylation may contribute to inflammatory disorders. The regulation of genes like Mmp10 and Mmp13 by acetylated NF-κB suggests potential roles in tissue remodeling and inflammatory pathologies .

• Cancer biology: NF-κB acetylation affects genes involved in cell survival, proliferation, and immune evasion. The potential regulation of these processes through K122 acetylation may have implications for cancer development and progression.

• Immune dysfunction: The differential regulation of immune-related genes shown in the expression profiles (Tables 1 and 2) suggests that acetylation status influences immune responses . Abnormal K122 acetylation might contribute to immunological disorders.

• Neurodegenerative diseases: NF-κB plays important roles in neuroinflammation. The detection of acetylated RELA in mouse brain cells suggests potential relevance to neurological conditions where inflammation is a component.

• Therapeutic targeting potential: Understanding the specific effects of K122 acetylation could lead to more precise therapeutic strategies that target this specific modification rather than broadly inhibiting NF-κB, potentially reducing side effects while maintaining efficacy.

Future research using the Acetyl-RELA (K122) Antibody in disease models and patient samples could directly test these hypothetical connections and potentially reveal new therapeutic opportunities.