KAT2A Antibody

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

KAT2A is a histone acetyltransferase that plays essential roles in regulating gene expression through epigenetic modification. It functions primarily by acetylating histones, particularly H3K9, which typically leads to transcriptional activation. KAT2A is expressed in undifferentiated basal cells and spinous keratin 10-positive cells in the suprabasal layer of the epidermis, with weak to no detection in terminally differentiating cells . This expression pattern suggests a critical role in maintaining the undifferentiated state.

The significance of KAT2A in epigenetic research stems from its fundamental involvement in stem cell pluripotency and differentiation processes. Multiple in vitro studies have demonstrated that KAT2A regulates stem cell pluripotency, as evidenced by the fact that Kat2a-null mouse embryoid bodies were smaller with disorganized epiblasts and showed enhanced rates of myogenic differentiation . Furthermore, chemical inhibition of KAT2A in mouse embryonic stem cells reduced pluripotency and accelerated mesendodermal differentiation . These findings make KAT2A antibodies valuable tools for investigating epigenetic mechanisms controlling cellular identity and differentiation.

How do KAT2A and KAT2B differ functionally, and why might researchers need antibodies specific to each?

Despite being structurally similar paralogs, KAT2A and KAT2B exhibit divergent expression profiles and functions in cellular processes, particularly in epidermal homeostasis. Research has revealed that:

Researchers need specific antibodies for each protein to properly investigate their distinct roles in various cellular contexts. Using a specific KAT2A antibody is essential when studying processes like stem cell maintenance, while KAT2B-specific antibodies are more relevant for differentiation studies. The compensatory relationship between these proteins (KAT2A levels were significantly increased in Kat2b −/− keratinocytes) further necessitates specific antibodies to accurately distinguish their individual contributions .

What validation methods should researchers use to confirm KAT2A antibody specificity?

Proper validation of KAT2A antibodies is crucial for ensuring experimental reliability. Researchers should implement the following comprehensive validation strategy:

Primary Validation Methods:

• Genetic knockout/knockdown controls: Validate antibody specificity using KAT2A knockdown cell lines. This approach was effectively demonstrated in studies where stable knockdown cell lines were generated using lentiviral transduction with pLenti constructs expressing shRNA sequences against KAT2A . The absence of signal in these knockdown models confirms antibody specificity.

• Western blot analysis: Perform western blotting to verify that the antibody detects a protein of the expected molecular weight (~90 kDa for KAT2A). Multiple studies have successfully used western blotting to detect KAT2A, including analyses of protein levels following gene knockdown .

• Immunohistochemistry with proper controls: When performing IHC, include positive control tissues known to express KAT2A (such as epidermis basal layer) and negative controls (either tissues known not to express KAT2A or primary antibody omission controls) .

Advanced Validation Approaches:

• Expression correlation: Compare protein detection with mRNA expression data from qRT-PCR. For example, researchers have used qRT-PCR primers (forward 5-CCCGCTACGAAACCACTCAT-3, reverse 5-GCATGGACAGGAATTTGGGGA-3) to validate KAT2A expression levels .

• Multiple antibody concordance: Use at least two antibodies targeting different epitopes of KAT2A and confirm concordant results. Commercial antibodies like those targeting epitopes within 15 amino acids from the C-terminal half can be compared with antibodies targeting other regions .

Implementing these validation methods ensures that experimental results accurately reflect KAT2A biology rather than artifacts from non-specific antibody binding.

What are the optimal protocols for detecting KAT2A using Western blotting?

Western blotting is a fundamental technique for KAT2A protein detection and quantification. Based on published research methodologies, the following optimized protocol is recommended:

Sample Preparation:

• Extract total protein using standard cell lysis buffers containing protease inhibitors to prevent KAT2A degradation.

• Quantify protein concentration using Bradford or BCA assays to ensure equal loading.

• Denature 20-40 μg of protein sample in Laemmli buffer at 95°C for 5 minutes.

Gel Electrophoresis and Transfer:

• Separate proteins on 8-10% SDS-PAGE gels (KAT2A is approximately 90 kDa).

• Transfer to PVDF membranes using wet transfer at 100V for 90 minutes at 4°C.

• Verify transfer efficiency with Ponceau S staining.

Antibody Incubation:

• Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature.

• Incubate with primary KAT2A antibody (such as 66575-1-Ig, Proteintech) at 1:1000 dilution in 5% BSA/TBST overnight at 4°C .

• Wash three times (5 minutes each) with TBST.

• Incubate with appropriate HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature.

• Wash three times (10 minutes each) with TBST.

Detection and Normalization:

• Develop using ECL substrate and image using a digital imaging system.

• Use GAPDH as an internal reference for normalization .

Troubleshooting Tips:

• If KAT2A signal is weak, extend primary antibody incubation to 48 hours at 4°C.

• For tissues with low KAT2A expression, increase protein loading to 50-60 μg.

• If experiencing non-specific bands, increase blocking time to 2 hours and use more stringent washing conditions.

This protocol has been successfully implemented in multiple studies investigating KAT2A function in cellular differentiation and proliferation contexts .

How should researchers optimize immunohistochemistry protocols for KAT2A detection in different tissue types?

Optimizing immunohistochemistry (IHC) for KAT2A requires careful consideration of tissue-specific factors. Based on successful applications in skin tissue research, the following optimized protocol is recommended:

Tissue Preparation:

• Fix tissues in 4% paraformaldehyde for 24 hours at room temperature.

• Process tissues through graded alcohols and xylene.

• Embed in paraffin and section at 5 μm thickness.

Antigen Retrieval (Critical for KAT2A Detection):

• Deparaffinize sections through xylene and rehydrate through graded alcohols.

• Perform heat-induced epitope retrieval using sodium citrate buffer (pH 6.0) at 95°C for 20 minutes.

• Allow slides to cool to room temperature for 20 minutes before proceeding.

Antibody Incubation Parameters:

• Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes.

• Apply protein block (5% normal goat serum) for 1 hour at room temperature.

• Incubate with KAT2A primary antibody (verified for paraffin sections ) at 1:100-1:200 dilution overnight at 4°C.

• Wash thoroughly with PBS (3 × 5 minutes).

• Apply appropriate secondary antibody for 1 hour at room temperature.

• Wash thoroughly with PBS (3 × 5 minutes).

• Develop using DAB and counterstain with hematoxylin.

Tissue-Specific Considerations:

• Skin tissues: In human epidermis, KAT2A shows gradient expression with stronger signals in basal and spinous layers . Apply shorter DAB development times (2-3 minutes) to better visualize this gradient.

• Highly vascularized tissues: Extended blocking step (2 hours) may help reduce background staining.

• Neural tissues: Addition of 0.1% Triton X-100 to antibody diluent may improve penetration.

Controls and Validation:

• Include positive control tissues known to express KAT2A (epidermis basal cells).

• Include negative controls by omitting primary antibody.

• Consider using KAT2A knockdown tissue sections as additional specificity controls where available.

This protocol has been effective for detecting KAT2A in epidermal tissues, allowing researchers to visualize its differential expression across differentiation states in the epidermis .

What experimental approaches help distinguish between KAT2A and KAT2B in research studies?

Distinguishing between KAT2A and KAT2B is critical for accurately characterizing their distinct functions. The following experimental approaches are recommended:

Selective Genetic Manipulation:

• RNA interference: Use of specific shRNA sequences targeting either KAT2A or KAT2B as demonstrated in published studies. Lentiviral transduction with pLenti constructs expressing shRNA sequences has proven effective for creating stable knockdown cell lines .

• CRISPR-Cas9 knockout: Generate specific KAT2A or KAT2B knockout cell lines using CRISPR-Cas9, being careful to target non-homologous regions.

Expression Analysis at Multiple Levels:

• Protein detection: Use paralog-specific antibodies in western blotting with validated controls. For KAT2A, antibodies like 66575-1-Ig (Proteintech) have been successfully used .

• Transcript analysis: Employ qRT-PCR with paralog-specific primers. For KAT2A detection, primers (forward 5-CCCGCTACGAAACCACTCAT-3, reverse 5-GCATGGACAGGAATTTGGGGA-3) have been validated .

• In situ hybridization: This technique was successfully used to detect KAT2B mRNA in tissue sections when antibody specificity was a concern .

Functional Discrimination:

• Differentiation assays: KAT2A depletion leads to premature expression of epidermal differentiation genes, whereas KAT2B loss delays differentiation . Researchers can use this functional divergence to confirm the identity of each paralog.

• Rescue experiments: Perform rescue experiments by expressing shRNA-resistant constructs of either KAT2A or KAT2B in knockdown cells. This approach has been used to validate the specificity of observed phenotypes .

Mouse Models:

• Paralog-specific knockout models: Use available Kat2b knockout mice for in vivo studies, as Kat2b-null mice are viable, while Kat2a-null mice are embryonically lethal .

• Conditional knockout approaches: For studies requiring KAT2A deletion, conditional knockout approaches using tissue-specific Cre recombinase expression (such as K14-Cre for epidermis) can overcome the embryonic lethality issue .

These approaches collectively provide a robust framework for distinguishing between these paralogs while accounting for their compensatory relationship, as evidenced by increased KAT2A levels in Kat2b−/− keratinocytes .

How can researchers use KAT2A antibodies to investigate its role in cancer biomarker studies?

KAT2A has emerged as a potential biomarker in cancer research, particularly in diffuse large B-cell lymphoma (DLBCL). The following methodological approach outlines how researchers can effectively use KAT2A antibodies in cancer biomarker studies:

Functional Validation in Cancer Cell Lines:

• Knockdown studies: Use siRNA or shRNA to deplete KAT2A in cancer cell lines and evaluate effects on:

  • Cell proliferation using CCK-8 assays at 24, 48, and 72 hours post-transfection

  • Cell cycle progression using flow cytometry with DNA staining

  • Apoptosis using Annexin V-PE/7-AAD staining and flow cytometry

Molecular Mechanism Investigation:

• ChIP-seq analysis: Use ChIP-grade KAT2A antibodies to identify genomic binding sites and correlate with gene expression changes in cancer cells.

• RNA-seq following KAT2A modulation: Prepare RNA-seq libraries from polyA-selected RNA (100 ng) using techniques like the NEBNext Ultra II Directional RNA Library Prep Kit, with sequencing depth over 29 million mappable reads .

• Pathway analysis: Apply Gene Ontology enrichment (GO) and Gene Set Enrichment Analysis (GSEA) to identify KAT2A-regulated cancer-relevant pathways .

Biomarker Validation:

• Multi-cohort validation: Evaluate KAT2A expression across independent patient cohorts, such as those from GEO database (GSE10846 and GSE31312) and TCGA-DLBC .

• Multivariate analysis: Perform multivariate analysis to determine whether KAT2A expression is an independent prognostic factor after adjusting for established clinical variables.

• Cutpoint determination: Use statistical tools like the "surv-cutpoint" function in R to determine optimal cutoff points for stratifying patients into high- and low-KAT2A expression groups .

This methodological framework has been applied successfully in DLBCL research, where KAT2A was identified as a potential biomarker related to immune infiltration and malignant pathways, with distinct tumor immune microenvironments and prognoses associated with expression levels .

What controls are essential when using KAT2A antibodies to study epidermal differentiation?

When investigating KAT2A's role in epidermal differentiation, proper controls are essential for generating reliable and interpretable data. Based on published research protocols, the following controls should be implemented:

Genetic Controls:

• KAT2A knockdown/knockout: Include keratinocytes with stable KAT2A knockdown using validated shRNA sequences. This control is critical for confirming antibody specificity and phenotypic effects .

• KAT2B knockdown comparison: Include parallel KAT2B knockdown samples to distinguish paralog-specific effects, as these proteins have divergent functions in epidermal differentiation .

• Rescue controls: Include cells expressing shRNA-resistant KAT2A constructs to confirm phenotype specificity. This approach has been used with shRNA-resistant full-length KAT2A CDS subcloned into pLenti-P2A-Bsd vectors .

Functional Domain Controls:

• Catalytic mutant: Include the mAT KAT2A mutant (generated by swapping guanine and adenine at bases 1723–1724) to determine which effects depend on KAT2A's acetyltransferase activity .

• Domain deletion mutants: Include additional functional mutants such as delBr KAT2A (bases 1–2169) and delN KAT2A (bases 1084–2514) to dissect domain-specific functions .

Differentiation Stage Controls:

• Time course samples: Collect cells at multiple differentiation timepoints (day 0, undifferentiated; days 3-6, differentiating) to track KAT2A expression changes throughout differentiation .

• Differentiation markers: Include parallel detection of established differentiation markers (e.g., KRT10) to correlate KAT2A expression with known differentiation stages .

Technical Controls:

• Antibody validation controls: Include isotype controls and primary antibody omission controls in all immunological assays.

• Reference gene controls: For qPCR analysis, normalize target Ct values to established reference genes like RPL13A, and calculate relative fold changes using the 2−ΔΔCt method .

In Vivo Controls:

• Wild-type comparison: When using Kat2b knockout mice, include age-matched wild-type mice as controls .

• Heterozygote controls: Include heterozygotes (which contained similar KAT2B levels as wild-type mice) as additional controls .

These comprehensive controls have been effectively implemented in epidermal differentiation research, revealing that KAT2A is enriched in undifferentiated keratinocytes where it functions via its HAT activity to maintain the self-renewal state and balance the pro-differentiation functions of KAT2B .

How can researchers troubleshoot non-specific binding issues with KAT2A antibodies?

Non-specific binding is a common challenge when working with KAT2A antibodies. The following troubleshooting strategies address specific issues that may arise:

Multiple Bands in Western Blotting:

• Increase blocking stringency: Extend blocking time to 2 hours using 5% BSA in TBST rather than non-fat milk.

• Optimize antibody dilution: Test a dilution series (1:500, 1:1000, 1:2000) to identify optimal concentration that maintains specific signal while reducing background.

• Use freshly prepared samples: KAT2A may be subject to proteolytic degradation; ensure samples contain sufficient protease inhibitors and are freshly prepared.

• Validate with knockout controls: Compare bands with KAT2A knockdown samples to identify which band represents genuine KAT2A signal .

Background in Immunohistochemistry:

• Optimize antigen retrieval: Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA pH 8.0, enzymatic retrieval) to identify optimal protocol for your tissue type.

• Titrate primary antibody: Perform a dilution series (1:50, 1:100, 1:200, 1:500) to determine optimal concentration.

• Increase washing steps: Add additional washing steps (5 × 5 minutes) with 0.1% Tween-20 in PBS.

• Use alternative detection systems: If using HRP-based detection, consider more sensitive detection systems with lower background (e.g., polymer-based detection systems).

Cross-reactivity with KAT2B:

• Epitope selection: Choose antibodies targeting non-conserved regions between KAT2A and KAT2B.

• Validation with paralog-specific controls: Test antibody reactivity in cells overexpressing either KAT2A or KAT2B.

• Alternative detection methods: Consider using mRNA-based detection methods like in situ hybridization, which was successfully used for KAT2B detection when antibody specificity was problematic .

Inconsistent Results Across Experiments:

• Standardize cell culture conditions: KAT2A expression can vary with cell density and differentiation state in keratinocytes. Maintain consistent culture conditions (70% confluency for NTERTs and 100% confluency for NHEKs before differentiation) .

• Control for differentiation state: KAT2A and KAT2B show differentiation-dependent expression patterns, so ensure cells are at equivalent differentiation stages across experiments .

• Use positive control samples: Include a standard positive control sample across all experiments to normalize for inter-experimental variation.

These troubleshooting approaches are based on established protocols that have successfully detected KAT2A in various experimental contexts, including challenging differentiation models and in vivo systems .

What cutting-edge applications are emerging for KAT2A antibodies in multi-omics research?

KAT2A antibodies are increasingly being integrated into cutting-edge multi-omics approaches, opening new frontiers in epigenetic research. The following emerging applications represent the leading edge of KAT2A research:

Integrative Epigenomic Profiling:

• ChIP-sequencing combined with RNA-seq: This approach enables correlation between KAT2A genomic binding sites and transcriptional changes. RNA-seq libraries can be prepared from polyA-selected RNA using techniques like the NEBNext Ultra II Directional RNA Library Prep Kit, with sequencing depth over 29 million mappable reads .

• CUT&RUN and CUT&Tag: These techniques offer higher resolution and lower background than traditional ChIP, requiring fewer cells and less antibody, making them ideal for precious clinical samples.

• Single-cell approaches: Emerging single-cell ChIP-seq and CUT&Tag protocols allow investigation of KAT2A binding heterogeneity within tissues.

Spatial Multi-omics Applications:

• Imaging mass cytometry: Combining KAT2A antibodies with metal-tagged antibodies against other epigenetic marks and cell-type markers allows simultaneous visualization of multiple proteins in tissue sections at subcellular resolution.

• Spatial transcriptomics integration: Correlating KAT2A protein localization from immunofluorescence with spatial transcriptomics data provides insights into region-specific gene regulation.

Functional Genomics Screens:

• CRISPR-based epigenome editing: KAT2A antibodies are being used to validate the effects of CRISPR-mediated recruitment of KAT2A to specific genomic loci.

• Proximity labeling approaches: BioID or APEX2 fused to KAT2A combined with antibody validation is revealing novel protein interaction networks in different cellular contexts.

Therapeutic Target Validation:

• Patient-derived organoids: KAT2A antibodies are being used to assess expression in 3D organoid models, providing insights into its role in development and disease.

• Pharmacological inhibitor studies: Antibodies are essential for validating the effects of emerging small molecule inhibitors of KAT2A in various disease models.

Biomarker Development in Precision Medicine:

• Multiplexed tissue imaging: Combining KAT2A antibodies with other biomarkers in multiplexed immunofluorescence panels is helping stratify patients for clinical trials.

• Liquid biopsy applications: Detection of KAT2A in circulating tumor cells or exosomes using sensitive antibody-based capture methods is being explored as a non-invasive biomarker approach.

These advanced applications build upon the foundational research showing KAT2A's role in maintaining the undifferentiated state in keratinocytes and its potential as a biomarker in DLBCL . The integration of KAT2A antibodies into multi-omics approaches is revealing unprecedented insights into the mechanisms by which this histone acetyltransferase regulates cell fate decisions and contributes to disease processes.