CAF1-3 Antibody

What are the main types of CAF1 antibodies available for research?

Researchers primarily use antibodies targeting specific CAF1 subunits rather than the entire complex. Common antibodies include:

• Anti-CHAF1A (p150) antibodies such as SS48

• Anti-CHAF1B (p60) antibodies including SS53 and SS96

• Anti-RBBP4 (p48) antibodies

• Antibodies against phosphorylated forms of CAF1 subunits

It's important to note that the term "CAF1-3" can also refer to a specific monoclonal antibody (CaF1-5C3, also known as CAF3-5C3 or CAF3) that targets ATP2A1 (calcium ATPase), which is distinct from the CAF1 histone chaperone complex .

What are the recommended applications for CAF1 antibodies in research?

CAF1 antibodies are valuable tools for:

• Western blot analysis of CAF1 subunit expression

• Immunoprecipitation to study CAF1 interactions with binding partners

• Chromatin immunoprecipitation (ChIP) to study CAF1 recruitment to specific genomic loci

• Immunofluorescence to visualize CAF1 localization, particularly at sites of DNA damage

Application-specific dilutions typically range from 1:1000 for p150 detection to 1:5000 for p60 detection in immunoblots .

How should I optimize immunoblot protocols for CAF1 antibodies?

For optimal results when using CAF1 antibodies in immunoblots:

• Sample preparation:

  • Prepare total cell extracts or pulldown samples by SDS-PAGE

  • Transfer proteins to nitrocellulose membranes

• Recommended antibody dilutions:

  • CAF1 p150 (SS48): 1:1000

  • CAF1 p60 (SS53 or SS53/SS96 mixture): 1:5000

• Detection optimization:

  • Block with 5% non-fat milk in TBST

  • Incubate with primary antibody overnight at 4°C

  • Use HRP-conjugated secondary antibodies and enhance sensitivity with ECL detection

A critical consideration is that different antibodies may recognize specific forms of CAF1 subunits, including full-length and truncated variants, so molecular weight should be carefully assessed .

What are the best protocols for studying CAF1 recruitment to DNA damage sites?

To study CAF1 recruitment to DNA damage sites:

• Localized UV irradiation method:

  • Apply UV irradiation through filters to create localized DNA damage

  • Fix cells 10-30 minutes post-irradiation

  • Perform detergent extraction before fixation to remove soluble nuclear proteins

  • Use antibodies against both p60 and p150 subunits to confirm complete CAF1 recruitment

• Key experimental parameters:

  • UV dose: 50-150 J/m² shows optimal visualization

  • Timing: Recruitment is detectable within 10 minutes post-irradiation

  • Co-staining: Include PCNA antibodies as a damage site marker

For reliable detection, use at least two independent antibodies targeting different CAF1 subunits, as p60 and p150 recruitment is tightly coupled .

How can I set up ChIP experiments to study CAF1's role in histone deposition?

For effective ChIP experiments investigating CAF1's role in histone deposition:

• Preparation steps:

  • Establish cell lines expressing HA-tagged histone variants (H3.1/H3.3) if studying histone variant deposition

  • Consider using CRISPR/Cas9 to deplete CAF1 subunits (e.g., CHAF1B)

• ChIP protocol adjustments:

  • Cross-link with formaldehyde (1%) for 10 minutes

  • Sonicate to generate fragments of 200-500 bp

  • Immunoprecipitate with antibodies against histone variants or their epitope tags

  • For DNA damage studies, consider treating cells with acyclovir to prevent lytic genome production

• Controls to include:

  • IgG control antibodies

  • Input DNA samples

  • Host genome loci as internal controls

When analyzing data, normalize ChIP signals to account for differences in histone levels due to CAF1 depletion effects .

How can I investigate CAF1's role in establishing and maintaining viral latency?

To investigate CAF1's role in viral latency (using EBV as a model system):

• Experimental approaches:

  • CRISPR/Cas9 targeting of CAF1 subunits (CHAF1A, CHAF1B, RBBP4)

  • Stable knockdown using shRNAs

  • Rescue experiments with PAM site mutant cDNAs

• Readouts for viral latency disruption:

  • Monitor viral protein expression (e.g., gp350 for EBV)

  • Quantify viral genome copy number in supernatants (encapsidated DNA)

  • Measure expression of viral latency-associated transcripts using RNAseq

• Mechanistic investigations:

  • ChIP-qPCR to assess histone H3.1 and H3.3 occupancy at viral promoters

  • Monitor repressive histone marks (H3K9me3, H3K27me3) at key viral genomic sites

Research has shown that CAF1 depletion significantly reduces H3.1 and H3.3 occupancy at viral genomic sites including lytic gene promoters and replication origins, leading to viral reactivation from latency .

How does CAF1 coordinate with other histone chaperones in replication-dependent and independent pathways?

To study the coordination between CAF1 and other histone chaperones:

• Experimental strategies:

  • Co-immunoprecipitation to identify interacting partners

  • Sequential depletion experiments targeting multiple chaperones

  • Pulse-chase experiments with tagged histones

• Key interactions to investigate:

  • CAF1 and ASF1A-H3-H4 interactions

  • Coordination with HIRA for replication-independent deposition

  • Competition or cooperation with ATRX/DAXX

• Functional assays:

  • Measure histone loading kinetics during different cell cycle phases

  • Compare replication-dependent vs. independent pathways using cell synchronization

  • Analyze recovery after DNA damage in cells with single vs. combined chaperone deficiencies

Studies have shown that human ASF1A-H3-H4 interacts directly with CAF1, and this interaction is important for proper nucleosome assembly during DNA replication .

What are the differences in CAF1 function between leading and lagging strand DNA replication?

Recent research has revealed distinct CAF1 mechanisms on leading versus lagging DNA strands:

• Experimental setup to investigate strand-specific functions:

  • Reconstituted in vitro replication systems

  • Strand-specific labeling techniques

  • Single-molecule approaches

• Key findings on differential mechanisms:

  • DNA and histones promote CAF1 recruitment to PCNA

  • Two CAF1 complexes are required for efficient nucleosome assembly

  • CAF1 competes differently with replisome components depending on the strand

• Technical approaches for strand discrimination:

  • Modified DNA templates with strand-specific markers

  • Replisome reconstitution with purified components

  • Strand-specific ChIP-seq approaches

This strand-specific function of CAF1 has important implications for understanding how chromatin is assembled during DNA replication and how epigenetic information might be maintained differently on leading versus lagging strands .

How can I resolve common issues with CAF1 antibody specificity in different applications?

When encountering specificity issues with CAF1 antibodies:

• Validation approaches:

  • Use CRISPR/Cas9 knockout cells as negative controls

  • Perform peptide competition assays

  • Compare results from multiple antibodies targeting different epitopes

  • Include positive controls with overexpressed tagged proteins

• Common issues and solutions:

  Issue	Potential Cause	Solution
  Multiple bands in Western blot	Degradation products or isoforms	Use fresh samples with protease inhibitors; validate with knockout controls
  Weak or no signal	Low target expression or epitope masking	Increase antibody concentration; try alternative antibodies or epitope retrieval
  High background	Non-specific binding	Increase blocking time; use more stringent washes; titrate antibody
  Inconsistent results	Lot-to-lot variation	Validate each new lot; consider monoclonal antibodies for consistency

• Application-specific considerations:

  • For ChIP, increase chromatin fragmentation efficiency

  • For immunofluorescence, optimize fixation methods (some epitopes are sensitive to certain fixatives)

How should I interpret changes in histone loading patterns after CAF1 depletion?

When analyzing histone loading patterns after CAF1 depletion:

• Data normalization considerations:

  • Account for potential changes in total histone levels

  • Consider cell cycle effects (CAF1 depletion may alter cell cycle distribution)

  • Use spike-in controls for quantitative ChIP experiments

• Distinguishing direct from indirect effects:

  • Examine short-term vs. long-term depletion effects

  • Consider compensatory mechanisms (e.g., upregulation of HIRA)

  • Analyze effects on replication-dependent (H3.1) and independent (H3.3) histone variants

• Interpreting genomic location-specific effects:

  • Compare euchromatic vs. heterochromatic regions

  • Analyze replication timing domains

  • Consider chromatin domain boundaries and insulators

Research shows that CAF1 depletion reduces both H3.1 and H3.3 levels at specific genomic sites, despite not reducing steady-state levels of these histones, suggesting redistribution rather than degradation .

What controls are essential when studying CAF1 protein-protein interactions?

When investigating CAF1 protein-protein interactions:

• Critical controls for pull-down experiments:

  • Use tag-only vectors as negative controls

  • Include reciprocal IP experiments (pull down partner A to detect B, then pull down B to detect A)

  • Test interactions in both native conditions and after cross-linking

  • Include non-interacting proteins as negative controls

• Validating direct interactions:

  • Use recombinant proteins for in vitro binding assays

  • Perform domain mapping to identify interaction regions

  • Consider mutational analysis of key residues

• Analyzing interaction dynamics:

  • Test interactions under different cellular conditions (e.g., DNA damage, cell cycle phases)

  • Use proximity ligation assays or FRET to confirm interactions in cells

  • Consider stability of complexes under different salt or detergent conditions

Studies have demonstrated that the Cac1 subunit of CAF-1 functions as a scaffold within the CAF-1-H3/H4 complex, with key cross-links from Cac1 to both Cac2 and Cac3, while no direct interactions between Cac2 and Cac3 were observed .

How is CAF1 involved in regulating cellular identity and gene expression programs?

CAF1's role in cellular identity regulation represents an emerging research area:

• Experimental approaches to study identity regulation:

  • Genome-wide CAF1 binding profiles in different cell types

  • Effects of CAF1 depletion on cell type-specific gene expression

  • Integration with chromatin accessibility and histone modification data

• Key findings from current research:

  • CAF1 depletion can induce expression of genes typically silenced in specific cell types

  • CAF1 cooperates with DNA methyltransferases to maintain silencing

  • CAF1 has roles in heterochromatin organization that affect cell identity

• Technical considerations:

  • Use inducible depletion systems to avoid cell cycle arrest

  • Distinguish between direct and indirect effects through time-course experiments

  • Consider compensatory mechanisms that may mask phenotypes

Research has shown that CAF1 deficiency can induce gene expression (e.g., Cd4 in CD8+ T cells) without significant changes in DNA methylation or chromatin accessibility, suggesting complex regulatory mechanisms .

What are the latest methods for studying CAF1 dynamics during DNA replication and repair?

Cutting-edge approaches for studying CAF1 dynamics include:

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize CAF1 at replication forks

  • Live-cell imaging with fluorescently tagged CAF1 subunits

  • Single-molecule tracking to follow CAF1 movement

• Biochemical reconstitution approaches:

  • Reconstituted replication systems with defined components

  • Single-molecule approaches to study CAF1 loading kinetics

  • Microfluidic systems to control DNA damage induction

• Genomic mapping strategies:

  • iPOND (isolation of Proteins On Nascent DNA) coupled with mass spectrometry

  • ChIP-seq with nascent DNA labeling

  • Strand-specific mapping of histone variants

Recent studies have shown that CAF1 is recruited to DNA damage sites within 10 minutes post-irradiation, and this recruitment follows a dose-dependent pattern that correlates with damage intensity .

How might targeting CAF1 function be leveraged for therapeutic applications?

Potential therapeutic applications targeting CAF1 function:

• Viral latency disruption strategies:

  • Small molecule inhibitors of CAF1-histone interactions

  • Peptide inhibitors targeting CAF1 complex formation

  • Approaches to disrupt CAF1 recruitment to viral genomes

• Cancer therapeutic approaches:

  • Synthetic lethality with DNA repair pathways

  • Disruption of CAF1-dependent silencing in cancer cells

  • Combination with epigenetic modulators

• Experimental considerations for drug development:

  • Target specificity (discriminating between CAF1 and other histone chaperones)

  • Cell type-specific effects

  • Potential for resistance mechanisms

Research indicates that targeting CAF1 could potentially reactivate latent viruses like EBV, which could be leveraged for "shock and kill" therapeutic strategies against latent viral infections .

What CAF1 genetic tools and model systems are most valuable for mechanistic studies?

Recommended genetic tools and models for CAF1 research:

• Cell line models with validated modifications:

  • CRISPR/Cas9 knockout or knockdown systems for CAF1 subunits

  • Cell lines expressing tagged versions of CAF1 components

  • Inducible depletion systems to avoid cell cycle effects

• Viral model systems:

  • EBV+ Akata cells for viral latency studies

  • Cell lines with stable HA-tagged histone variant expression

• Recombinant protein resources:

  • Purification protocols for recombinant CAF1 subunits

  • ASF1A-H3-H4 complexes for interaction studies

  • Modified histones for specificity studies

Several studies have successfully used EBV+ Akata cells with CRISPR/Cas9-mediated depletion of CHAF1B combined with ChIP-qPCR to study CAF1's role in viral latency maintenance .

What are the key techniques for analyzing CAF1's role in nucleosome assembly?

Essential techniques for studying CAF1's nucleosome assembly functions:

• Biochemical assembly assays:

  • In vitro nucleosome assembly reactions with purified components

  • Micrococcal nuclease digestion patterns to assess nucleosome spacing

  • Supercoiling assays to measure nucleosome formation

• Structural biology approaches:

  • Hydrogen/deuterium exchange mass spectrometry

  • Chemical cross-linking coupled to mass spectrometry

  • Cryo-EM analysis of CAF1-histone complexes

• Functional assays:

  • Analysis of histone variant incorporation at specific genomic loci

  • Replisome reconstitution systems to study coordination with DNA synthesis

  • Pull-down assays to identify interactions between CAF1 components

Studies utilizing hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry have revealed that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex and can promote histone tetramerization independent of other subunits .