KAT14 Antibody

What is KAT14 and what are its primary cellular functions?

KAT14 functions as a component of the ATAC complex with histone acetyltransferase activity on histones H3 and H4. It serves as a scaffold that promotes ATAC complex stability while exhibiting weak intrinsic histone acetyltransferase activity toward histone H4. The protein is required for normal progression through both G1 and G2/M phases of the cell cycle . KAT14 is primarily localized in the nucleus, with some cytoplasmic expression also reported . The human KAT14 gene is located at chromosome position 20p11.23, and the protein has a molecular mass of approximately 88.844 kDa .

What types of KAT14 antibodies are currently available for research?

Several types of KAT14 antibodies are available for research purposes:

Polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide higher specificity for targeted applications . Recombinant antibodies represent the ultimate renewable reagent type with advantages in adaptability, such as the ability to switch IgG subclass or use molecular engineering to achieve higher affinity binding .

How should KAT14 antibodies be validated for research applications?

Optimal validation of KAT14 antibodies requires using both genetic and orthogonal approaches:

• Genetic approach: The gold standard for antibody validation involves comparing wild-type cells with isogenic CRISPR knockout (KO) versions of the same cell line. This method provides rigorous and broadly applicable results but comes at a higher cost .

• Orthogonal approach: This method correlates antibody results with known information about KAT14 protein characteristics, such as molecular weight, subcellular localization, or expression patterns. While more commonly used (61% of Western blot validations), this approach is less definitive than genetic validation methods .

For optimal experimental design, researchers should select cell lines with TPM+1 > 2 as an RNA-level threshold for creating CRISPR knockouts. Among commonly used cell lines showing adequate KAT14 expression, those with short doubling times that are amenable to CRISPR-Cas9 technology are preferred for validation studies .

What are the recommended protocols for using KAT14 antibodies in Western blotting?

For Western blotting applications with polyclonal KAT14 antibodies, the recommended dilution range is 1:500-1:2000 . The optimal protocol includes:

• Sample preparation: Extract proteins using a buffer containing phosphatase and protease inhibitors

• Protein separation: Use 8-12% SDS-PAGE gels (KAT14 molecular weight: ~88.8 kDa)

• Transfer: Transfer to PVDF or nitrocellulose membranes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Dilute KAT14 antibody in blocking buffer and incubate overnight at 4°C

• Secondary antibody: Anti-rabbit or anti-mouse HRP-conjugated antibody (depending on primary antibody host)

• Detection: Enhanced chemiluminescence (ECL) reagents

When validating KAT14 antibodies for Western blotting, 80-89% of antibodies recommended based on either orthogonal or genetic validation strategies successfully detected the intended target protein .

How can KAT14 antibodies be used to study the ATAC complex and histone modifications?

KAT14 antibodies can be employed to investigate ATAC complex dynamics through several methodological approaches:

• Co-immunoprecipitation (Co-IP): Use KAT14 antibodies to pull down the protein and associated complex members, followed by Western blotting or mass spectrometry to identify interacting partners.

• Chromatin Immunoprecipitation (ChIP): KAT14 antibodies can be used to identify genomic regions where the ATAC complex is bound, providing insights into its role in transcriptional regulation.

• Histone acetyltransferase assays: After immunoprecipitating KAT14, researchers can perform in vitro histone acetyltransferase assays to assess its enzymatic activity toward specific histone substrates, particularly H4 .

When designing these experiments, it's essential to include appropriate controls such as IgG controls for immunoprecipitation and knockout/knockdown controls to verify antibody specificity.

What considerations should be made when using KAT14 antibodies for immunofluorescence studies?

When using KAT14 antibodies for immunofluorescence applications, several methodological considerations are important:

• Fixation method: Paraformaldehyde (4%) is generally recommended, but methanol fixation may preserve certain epitopes better.

• Permeabilization: Use 0.1-0.5% Triton X-100 to facilitate antibody access to nuclear targets.

• Antibody validation: Only 38% of antibodies recommended based on orthogonal strategies for immunofluorescence were confirmed using KO cells as controls, indicating more rigorous validation is needed for this application .

• Co-localization studies: Consider dual staining with markers for nuclear structures or other ATAC complex components to confirm specificity and provide functional insights.

• Conjugated antibodies: For multi-color imaging or flow cytometry, directly conjugated antibodies (such as Alexa Fluor 488, 555, or 647 conjugates) may provide better resolution with less background .

How can non-specific binding be reduced when using KAT14 antibodies?

Non-specific binding is a common challenge when working with antibodies. For KAT14 antibodies, consider these methodological approaches:

• Optimization of blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers) and concentrations.

• Antibody dilution series: Test a range of dilutions to find the optimal signal-to-noise ratio. For ELISA applications, recommended dilutions range from 1:5000-1:10000, while Western blotting typically requires 1:500-1:2000 .

• Inclusion of genetic controls: When possible, include samples from KAT14 knockout or knockdown cells to identify non-specific bands or staining.

• Pre-adsorption: Consider pre-adsorbing the antibody with the immunizing peptide or with tissues/cells not expressing the target to reduce non-specific binding.

• Buffer optimization: Adjusting salt concentration and detergent levels can help reduce non-specific binding while preserving specific signals.

What are the advantages of using recombinant KAT14 antibodies compared to conventional antibodies?

Recombinant antibodies offer several methodological advantages in KAT14 research:

• Reproducibility: Recombinant antibodies provide batch-to-batch consistency, eliminating variability inherent in polyclonal antibodies.

• Adaptability: The molecular structure can be engineered to switch IgG subclass or achieve higher affinity binding than B-cell generated antibodies .

• Renewable source: Unlike hybridoma-derived monoclonal antibodies, recombinant antibodies can be produced indefinitely without relying on hybridoma stability.

• Defined specificity: The exact epitope and binding characteristics can be well-defined and optimized for specific applications.

• Reduced background: Recombinant antibodies often exhibit lower non-specific binding, particularly in complex assays like immunohistochemistry or immunofluorescence.

How might machine learning approaches improve KAT14 antibody research?

Recent developments in machine learning offer promising methodologies for KAT14 antibody research:

• Active learning for antibody-antigen binding prediction: Novel active learning strategies can improve experimental efficiency in library-on-library settings, potentially reducing the number of required antigen mutant variants by up to 35% .

• Prediction of antibody specificity: Machine learning models trained on many-to-many relationships between antibodies and antigens can help predict target binding, although challenges remain for out-of-distribution prediction scenarios .

• Optimization of validation protocols: Computational approaches can help design optimal validation experiments by identifying the most informative antibody-antigen pairs for testing.

These computational approaches could significantly reduce the cost and time required for KAT14 antibody validation while improving prediction accuracy of antibody performance across different applications.

What considerations should be made when studying KAT14's role in cell cycle regulation?

When investigating KAT14's role in cell cycle regulation, several methodological approaches are recommended:

• Synchronized cell populations: Use methods like double thymidine block or nocodazole treatment to synchronize cells at specific cell cycle phases to observe KAT14's dynamic behavior.

• Cell cycle markers: Co-stain with established cell cycle phase markers (cyclin D1 for G1, cyclin E for G1/S, cyclin A for S, cyclin B1 for G2/M) to correlate KAT14 activity with specific phases.

• KAT14 knockdown/knockout timing: Since KAT14 is required for normal progression through G1 and G2/M phases , design experiments with inducible knockdown/knockout systems to examine phase-specific effects.

• Histone modification analysis: Monitor changes in histone H3 and H4 acetylation patterns throughout the cell cycle using specific acetylation site antibodies in conjunction with KAT14 antibodies.

• ATAC complex dynamics: Investigate how the composition and activity of the ATAC complex changes during cell cycle progression using co-immunoprecipitation with KAT14 antibodies.