KAT2A Antibody, Biotin conjugated

What is KAT2A and why is it significant in epigenetic research?

KAT2A/GCN5 is a nuclear A-type histone acetyltransferase that directly impacts gene transcription. It plays crucial roles in cell cycle regulation, DNA replication, and DNA repair, making it a key player in maintaining genome stability . KAT2A functions as a coactivator of the c-MYC oncogene and demonstrates oncogenic roles in various cancers. Its significance in epigenetic research stems from its ability to act not only as an acetyltransferase but also as a glutaryltransferase, succinyltransferase, or malonyltransferase, depending on the cellular context . These diverse enzymatic capabilities allow KAT2A to regulate gene expression through multiple epigenetic modifications, contributing to various cellular processes including differentiation, proliferation, and cellular identity maintenance.

What are the recommended applications for KAT2A antibodies in standard research protocols?

Based on experimental validation data, KAT2A antibodies are suitable for multiple research applications with specific recommended dilutions:

Note: KAT2A antibodies may not be suitable for immunohistochemistry (IHC) testing in all contexts . Researchers should perform preliminary validation experiments to determine optimal conditions for their specific experimental system, as results may be sample-dependent.

How does biotin conjugation affect KAT2A antibody performance compared to unconjugated versions?

Biotin conjugation provides significant advantages in KAT2A detection through enhanced signal amplification. The biotin tag allows for secondary detection using streptavidin-conjugated reporters (fluorophores, enzymes, or gold particles), which can substantially increase sensitivity compared to unconjugated primary antibodies.

For optimal performance with biotin-conjugated KAT2A antibodies:

• Include appropriate blocking steps using biotin-free blocking agents to minimize background

• Consider implementing streptavidin-biotin amplification systems for low-abundance KAT2A detection

• Account for possible steric hindrance effects of the biotin moiety by adjusting incubation times

• Verify that biotin conjugation hasn't compromised the antibody's binding epitope through parallel validation with unconjugated antibodies

Note that while biotin conjugation enhances detection sensitivity, it may potentially interfere with certain binding epitopes or alter antibody stability during long-term storage compared to unconjugated versions.

How can KAT2A antibodies be used to investigate transcriptional noise in hematological malignancies?

Recent research has revealed KAT2A's critical role in controlling transcriptional noise and cellular heterogeneity in acute myeloid leukemia (AML) . To investigate this phenomenon using biotin-conjugated KAT2A antibodies:

• Single-cell chromatin profiling: Utilize biotin-conjugated KAT2A antibodies in CUT&Tag or CUT&RUN assays to assess KAT2A binding at specific promoters at single-cell resolution.

• Coupled transcriptomics approach: Combine chromatin immunoprecipitation (ChIP) using biotin-conjugated KAT2A antibodies with single-cell RNA sequencing to correlate KAT2A binding patterns with transcriptional heterogeneity.

• Transcriptional bursting analysis: Employ biotin-conjugated KAT2A antibodies in time-course experiments to capture dynamic KAT2A binding, followed by mathematical modeling of transcriptional bursting parameters.

Research has demonstrated that Kat2a loss specifically impacts transcriptional burst frequency in a subset of gene promoters, generating enhanced variability of transcript levels . This destabilization of target programs shifts leukemia cell fate from self-renewal to differentiation . Investigators can leverage biotin-conjugated KAT2A antibodies to further elucidate these mechanisms in various hematological malignancies beyond AML.

What controls should be implemented when using biotin-conjugated KAT2A antibodies in ChIP-seq experiments?

For rigorous ChIP-seq experiments using biotin-conjugated KAT2A antibodies, implement the following controls:

• Input control: Essential for normalizing sequencing depth and identifying enriched regions.

• IgG control: Use a biotin-conjugated isotype-matched IgG (IgG2a for mouse monoclonal antibodies) to establish background binding levels.

• KAT2A knockout/knockdown control: Include samples where KAT2A has been deleted or depleted to validate binding specificity.

• Blocking controls: Include biotin-blocking steps to prevent non-specific binding to endogenous biotinylated proteins.

• Spike-in normalization: Consider using spike-in chromatin from another species for quantitative comparisons between different conditions.

• Validation by orthogonal methods: Confirm key findings using unconjugated KAT2A antibodies or antibodies targeting KAT2A-associated histone modifications (H3K9ac).

When analyzing ChIP-seq data, focus on KAT2A-enriched genomic regions associated with H3K4me3 peaks (promoters) and H3K4me1 peaks (enhancers), as KAT2A has been shown to primarily function at these regulatory elements .

How can biotin-conjugated KAT2A antibodies be used to investigate DNA damage response mechanisms?

Recent research has identified a novel role for KAT2A in DNA damage response through its acetylation of PALB2 . To investigate this mechanism:

• Proximity ligation assays (PLA): Utilize biotin-conjugated KAT2A antibodies with antibodies against DNA repair proteins (PALB2, BRCA1/2, RAD51) to visualize and quantify protein-protein interactions before and after DNA damage induction.

• Sequential ChIP (ChIP-reChIP): Perform sequential immunoprecipitation with biotin-conjugated KAT2A antibodies followed by antibodies against DNA repair factors to identify genomic regions where these proteins co-localize.

• DNA damage kinetics: Track KAT2A binding to chromatin at different time points after DNA damage using biotin-conjugated antibodies combined with streptavidin pull-down assays.

Research has shown that KAT2A/2B acetylate a cluster of seven lysine residues (the 7K-patch) within the PALB2 chromatin association motif (ChAM), enhancing its direct association with nucleosomes . Following DNA damage, ChAM is rapidly deacetylated, increasing PALB2 mobility. These dynamics are critical for proper RAD51 foci formation in S phase and cell survival after DNA damage .

What are the optimal fixation and permeabilization conditions for detecting KAT2A using biotin-conjugated antibodies in immunofluorescence?

Optimal fixation and permeabilization conditions for KAT2A immunofluorescence detection:

When using SKOV-3 cells, which have been validated for KAT2A immunofluorescence , incubate with the biotin-conjugated KAT2A antibody overnight at 4°C for optimal signal-to-noise ratio. For quantitative analysis, include appropriate controls including non-specific biotin-conjugated antibodies and competitive binding controls with unconjugated antibodies.

How can reproducibility issues with KAT2A antibodies be addressed in multi-omics experiments?

To ensure reproducibility in multi-omics experiments using biotin-conjugated KAT2A antibodies:

• Antibody validation: Verify antibody specificity using Western blot against KAT2A knockout or knockdown samples before proceeding to more complex applications.

• Lot-to-lot consistency: Test different antibody lots side-by-side in pilot experiments to ensure consistent performance.

• Cross-platform validation: Compare results from biotin-conjugated antibodies with unconjugated versions in parallel experiments.

• Standard operating procedures: Develop and strictly adhere to detailed protocols for antibody handling, incubation, and washing steps.

• Technical replicates: Include multiple technical replicates to account for antibody binding variability.

• Reference standards: Include common reference samples across different experimental batches for normalization.

• Data normalization: Develop appropriate normalization strategies to account for technical variability in antibody performance.

For ChIP-seq specifically, ensure stringent quality control by assessing metrics like fraction of reads in peaks (FRiP), peak number consistency, and correlation between replicates.

What considerations should be made when using biotin-conjugated KAT2A antibodies to study its non-histone substrates?

When investigating KAT2A's interactions with non-histone substrates using biotin-conjugated antibodies:

• Crosslinking optimization: Standard formaldehyde crosslinking (used for histone interactions) may not efficiently capture transient interactions with non-histone substrates. Consider testing alternative crosslinkers like DSG (disuccinimidyl glutarate) or EGS (ethylene glycol bis(succinimidyl succinate)) for protein-protein interactions.

• Buffer modifications: Adjust immunoprecipitation buffers to preserve weaker or more transient interactions that may occur with non-histone substrates.

• Competition with endogenous biotin: Include additional blocking steps to prevent interference from endogenously biotinylated proteins, which are more abundant in the cytoplasm where many non-histone interactions occur.

• Mass spectrometry compatibility: When planning to identify novel substrates, ensure your biotin-conjugated antibody purification protocol is compatible with downstream mass spectrometry analysis.

Research has revealed that KAT2A can function as an acetyltransferase, glutaryltransferase, succinyltransferase, or malonyltransferase depending on the cellular context . Additionally, KAT2A/2B have been shown to acetylate non-histone proteins like PALB2, affecting DNA damage response pathways . When studying these diverse functions, researchers should design experiments that can distinguish between these different enzymatic activities.

How can background issues be minimized when using biotin-conjugated KAT2A antibodies in cells with high endogenous biotin?

To minimize background issues when using biotin-conjugated KAT2A antibodies:

• Pre-block endogenous biotin: Prior to antibody incubation, use commercial biotin/avidin blocking kits to mask endogenous biotin.

• Use appropriate secondary detection: For biotin-conjugated primary antibodies, employ streptavidin conjugates with minimal cross-reactivity to the experimental system.

• Optimize fixation: Over-fixation can increase autofluorescence and non-specific binding; titrate fixation times for your specific cell type.

• Include competing agents: Add free biotin in washing buffers to compete with weak non-specific interactions.

• Tissue-specific considerations: For tissues with naturally high biotin content (liver, kidney, brain), implement additional blocking steps and more stringent washing protocols.

For Western blot applications, consider adding avidin-agarose pre-clearing steps to your lysate preparation protocol to remove endogenously biotinylated proteins before separation and blotting.

What are the best approaches for quantifying KAT2A binding in relation to transcriptional bursting frequency?

To quantitatively assess KAT2A binding in relation to transcriptional bursting:

• Time-resolved ChIP-seq: Perform high-temporal resolution ChIP-seq with biotin-conjugated KAT2A antibodies to capture dynamic binding patterns.

• Nascent RNA sequencing: Combine KAT2A ChIP with nascent RNA sequencing techniques (such as NET-seq or GRO-seq) to correlate binding with active transcription.

• Mathematical modeling: Apply two-state models of gene expression to correlate KAT2A binding with burst frequency and burst size parameters.

• Single-molecule approaches: Use techniques like single-molecule RNA FISH combined with immunofluorescence to directly visualize the relationship between KAT2A binding and transcriptional output at the single-cell level.

Research has demonstrated that Kat2a loss specifically reduces transcriptional burst frequency in a subset of gene promoters, generating enhanced variability of transcript levels without affecting average expression levels . This finding highlights the importance of quantifying cell-to-cell variation rather than population averages when studying KAT2A function.

How can researchers address epitope masking issues when studying KAT2A in different protein complexes?

KAT2A functions in distinct protein complexes (SAGA and ATAC), which can lead to epitope masking issues when using antibodies. To address these challenges:

• Multiple antibody approach: Use multiple biotin-conjugated KAT2A antibodies targeting different epitopes to ensure detection regardless of complex formation.

• Pre-extraction protocols: Implement gentle pre-extraction steps to remove unbound or loosely bound KAT2A before fixation, revealing complex-bound epitopes.

• Native ChIP approach: Consider native (non-crosslinked) ChIP protocols that may better preserve certain protein-protein interactions.

• Complex-specific co-immunoprecipitation: Pair KAT2A immunoprecipitation with antibodies against known complex components (e.g., ATAC or SAGA complex members) to distinguish complex-specific functions.

• Mild sonication or nuclease treatment: Optimize chromatin fragmentation to preserve protein complexes while ensuring antibody accessibility.

Research shows KAT2A functions differently within SAGA versus ATAC complexes, with SAGA-associated KAT2A primarily mediating H3K9 acetylation and ATAC-associated KAT2A potentially affecting both promoters and enhancers . Consider these complex-specific functions when designing experiments and interpreting results.

How can biotin-conjugated KAT2A antibodies be utilized in studying leukemic stem cell dynamics?

To investigate leukemic stem cell (LSC) dynamics using biotin-conjugated KAT2A antibodies:

• Sequential ChIP-seq in sorted populations: Perform ChIP-seq with biotin-conjugated KAT2A antibodies on sorted LSC versus more differentiated leukemia cell populations to identify differential binding patterns.

• KAT2A tracking in differentiation assays: Use biotin-conjugated KAT2A antibodies to monitor changes in KAT2A localization during forced differentiation of leukemia cells.

• Genome-wide binding correlation with stemness markers: Integrate KAT2A ChIP-seq data with expression profiles of established LSC markers.

• Therapeutic response monitoring: Apply biotin-conjugated KAT2A antibodies to assess changes in chromatin regulation during response to differentiation-inducing therapies.

Research has demonstrated that loss of Kat2a delays leukemia development in mice and progressively depletes leukemia stem cells by causing more variability in gene expression . This leads to increased differentiation and decreased self-renewal. Biotin-conjugated KAT2A antibodies provide a sensitive tool to further elucidate how KAT2A stabilizes the leukemic stem cell state through epigenetic mechanisms.

What are the considerations for using biotin-conjugated KAT2A antibodies in multiplexed imaging approaches?

For multiplexed imaging with biotin-conjugated KAT2A antibodies:

• Sequential detection protocols: Develop protocols for sequential labeling, imaging, and signal removal when using multiple biotin-conjugated antibodies.

• Orthogonal detection systems: Combine biotin-streptavidin detection with other labeling approaches (e.g., direct fluorophore conjugation, HRP-based detection) to increase multiplexing capacity.

• Spatial resolution considerations: Account for the relatively large size of the biotin-streptavidin complex when analyzing co-localization at nanometer scale resolution.

• Signal amplification trade-offs: Balance the signal amplification advantages of biotin-conjugation against potential spatial resolution limitations.

• Cyclic immunofluorescence compatibility: Ensure biotin-streptavidin binding is effectively removed between cycles if implementing cyclic immunofluorescence protocols.

When studying KAT2A's interaction with DNA repair complexes after damage induction , multiplexed imaging can reveal spatial and temporal relationships between KAT2A and components like PALB2, BRCA1/2, and RAD51. Optimize antibody dilutions and detection reagents to achieve comparable signal intensities across targets for accurate co-localization analysis.

How does KAT2A acetylation activity influence nucleosome dynamics and chromatin accessibility?

To investigate KAT2A's impact on nucleosome dynamics and chromatin accessibility:

• Combined ChIP-ATAC approaches: Perform KAT2A ChIP followed by ATAC-seq on the immunoprecipitated chromatin to assess accessibility changes at KAT2A-bound regions.

• Nucleosome turnover assays: Use biotin-conjugated KAT2A antibodies alongside histone variant tracking (e.g., SNAP-tagged histones) to correlate KAT2A binding with nucleosome stability.

• In vitro reconstitution: Combine purified KAT2A with nucleosome substrates to directly assess acetylation-dependent changes in nucleosome structure and stability.

• Differential salt extraction: Implement chromatin fractionation with increasing salt concentrations to assess how KAT2A acetylation affects nucleosome stability in different genomic regions.

Research has shown that KAT2A's acetyltransferase activity influences transcription factor binding at promoters and that acetylation of non-histone proteins like PALB2 affects their chromatin association properties . These findings suggest KAT2A contributes to chromatin dynamics both through direct histone modification and through regulation of chromatin-binding proteins.