CAF1-2 Antibody

What is the structure of CAF-1 and which subunits can be targeted by antibodies?

CAF-1 is a heterotrimeric complex consisting of three essential subunits: p150 (CHAF1A), p60 (CHAF1B), and p48 (CHAF1C). Each subunit can be targeted by specific antibodies for research applications. The p150 and p60 subunits are most commonly targeted in experimental settings. The p150 subunit can be detected with antibodies such as SS48 or SS1, while p60 is typically detected with SS53 or a mixture of SS53 and SS96 monoclonal antibodies . The complex organization of these subunits is essential for CAF-1's function in histone deposition during DNA replication.

Why is CAF-1 critical for cellular function in human cells compared to yeast?

Unlike in yeast where CAF-1 is dispensable for cell viability, CAF-1 is essential in human cells for efficient progression through S-phase. Depletion of CAF-1 in human cell lines causes cells to accumulate in early and mid S-phase, replicate DNA slowly, and activate the checkpoint kinase Chk1 . This suggests that CAF-1 is necessary for coupling chromatin assembly with DNA replication in human cells, likely due to the increased complexity of human chromatin compared to yeast chromatin. The requirement for CAF-1 in human cells makes antibodies against its subunits valuable tools for studying replication-coupled chromatin assembly in higher eukaryotes.

How does CAF-1 interact with other chromatin assembly factors?

CAF-1 functions in coordination with other histone chaperones, notably ASF1A. Research has shown that purified yeast Caf1 and Asf1-H3-H4 interact with one another in solution . This interaction is part of a handoff mechanism where ASF1A presents H3-H4 dimers to CAF-1, which then deposits the histone tetramer (H3/H4)₂ onto newly synthesized DNA. Antibodies against both CAF-1 and ASF1A are therefore useful in co-immunoprecipitation experiments to study this interaction and the broader network of protein interactions in chromatin assembly pathways.

What are the optimal conditions for using CAF-1 antibodies in Western blot analysis?

For Western blot analysis of CAF-1 subunits, the following conditions have been empirically established:

Samples should be separated by SDS-PAGE (typically 10% gels) and transferred to nitrocellulose membranes. After blocking, membranes should be incubated with primary antibodies at the indicated dilutions overnight at 4°C, followed by appropriate secondary antibodies . This methodology provides reliable detection of CAF-1 subunits in various cell types and experimental conditions.

How can CAF-1 antibodies be used effectively in immunofluorescence studies?

For immunofluorescence analysis, cells should be plated on coverslips 16 hours before any experimental treatment. Following treatment, cells should be fixed in 2% paraformaldehyde in PBS and processed for immunofluorescence. To visualize chromatin-bound CAF-1, cells can be pre-extracted with Triton X-100 in CSK buffer, which removes soluble proteins but retains those bound to chromatin . Primary antibodies against CAF-1 p150 (SS1) can be used at 1:100 to 1:200 dilution, followed by appropriate fluorescent secondary antibodies (e.g., FITC anti-mouse). For co-localization studies with replication factors, dual staining with anti-PCNA (PC10) and anti-CAF-1 antibodies is particularly informative. Visualization should be performed using confocal microscopy or high-resolution fluorescence microscopy with oil-immersion objectives.

What is the methodology for performing immunoprecipitation with CAF-1 antibodies?

Immunoprecipitation of CAF-1 components can be performed from nuclear extracts or whole cell lysates depending on the experimental question. The following protocol has been shown to be effective:

• Prepare nuclear extracts from cells by hypotonic lysis followed by high-salt extraction of nuclear pellets.

• Incubate extracts with antibody-conjugated beads (e.g., SS24 anti-p60 beads or anti-HA 12CA5 beads for tagged versions) with rotation for 3 hours at 4°C.

• Wash the beads in Buffer A containing 100 mM salt.

• Elute bound proteins by boiling in SDS sample buffer or by specific peptide elution if preserving protein activity is required .

For cross-linking immunoprecipitation, dithiobis(succinimidyl propionate) can be used to stabilize protein-protein interactions before cell lysis, improving the detection of transient interactions with CAF-1 complexes .

How can researchers validate the specificity of CAF-1 antibodies?

Validating antibody specificity is crucial for reliable results. A comprehensive validation approach includes:

• Comparing antibody reactivity in control cells versus cells where CAF-1 subunits have been depleted via siRNA.

• Western blot analysis should show a significant reduction in band intensity corresponding to the targeted CAF-1 subunit in depleted cells.

• For immunofluorescence, comparing staining patterns in control versus CAF-1-depleted cells should show reduced or absent signal in depleted cells.

• Using recombinant CAF-1 proteins as positive controls in Western blots.

• Testing for cross-reactivity with related proteins, especially for p48 which shares homology with other proteins .

This multi-approach validation ensures that experimental observations are due to specific detection of CAF-1 rather than antibody cross-reactivity.

What are common issues when using CAF-1 antibodies and how can they be addressed?

Several technical challenges may arise when working with CAF-1 antibodies:

• High background in immunofluorescence: This can be reduced by increasing blocking time (2 hours in 5% BSA), using more stringent washing conditions, or titrating the antibody to determine optimal concentration.

• Weak signal in Western blots: Enriching for nuclear proteins can improve detection since CAF-1 is predominantly nuclear. Using phosphatase inhibitors (1 mM Na₃VO₄ and 10 mM NaF) in lysis buffers can also preserve phosphorylated forms that may affect antibody recognition .

• Multiple bands in Western blots: The p150 subunit may show multiple bands due to post-translational modifications or proteolytic processing. The SS48 antibody recognizes both full-length and truncated CAF-1, which can be advantageous for detecting different forms .

• Variable results in co-immunoprecipitation: Optimizing salt concentration in wash buffers is critical; typically, Buffer A with 100 mM salt provides a good balance between specificity and maintaining protein interactions .

How can CAF-1 antibodies be used to investigate replication-coupled chromatin assembly?

Investigating replication-coupled chromatin assembly requires sophisticated experimental approaches:

• In vitro chromatin assembly assays: Nuclear extracts from control and CAF-1-depleted cells can be prepared and tested for their ability to assemble chromatin on replicating DNA. The activity can be assessed through supercoiling assays or micrococcal nuclease digestion patterns. CAF-1 antibodies can be used to immunodeplete the complex from extracts to confirm its role in observed assembly activities .

• Pulse-chase experiments: Cells can be pulse-labeled with BrdU for short periods (10 min) to mark sites of active replication, followed by immunofluorescence with anti-BrdU and anti-CAF-1 antibodies to visualize co-localization at replication foci. This approach can reveal how CAF-1 dynamics correlate with DNA synthesis rates .

• Chromatin immunoprecipitation (ChIP): CAF-1 antibodies can be used in ChIP experiments to identify genomic regions where CAF-1 is actively involved in chromatin assembly during replication, providing insights into whether certain genomic regions depend more heavily on CAF-1 for proper chromatin structure.

What methods can be used to study CAF-1's role in maintaining cellular differentiation states?

Recent research has implicated CAF-1 in maintaining differentiated cell states. To investigate this aspect:

• siRNA depletion followed by transcriptome analysis: Compare gene expression profiles between control and CAF-1-depleted cells using RNA-seq. This can reveal genes whose expression depends on proper CAF-1 function .

• Chromatin accessibility assays: Combine CAF-1 depletion with ATAC-seq or DNase-seq to identify regions where chromatin structure changes upon CAF-1 loss, potentially explaining alterations in cell differentiation status.

• Cell fate marker analysis: Use immunofluorescence with antibodies against CAF-1 and cell-type-specific markers to track changes in cellular identity after CAF-1 manipulation.

• Sequential ChIP (Re-ChIP): To study CAF-1 interactions with lineage-specific transcription factors, perform ChIP with CAF-1 antibodies followed by a second round of immunoprecipitation with antibodies against relevant transcription factors .

How can researchers investigate the role of CAF-1 in cancer cell phenotypes?

CAF-1 has been implicated in cancer-related phenotypes such as increased cell motility and invasiveness. Advanced methods to study this connection include:

• Wound healing and invasion assays: Compare these phenotypes between control cells and those with modulated CAF-1 levels. In one study, CAF-1 depletion in untransformed MCF10A cells increased both cell motility in wound healing assays and cell invasion in Transwell Matrigel invasion assays, mimicking the effects of Src transformation .

• Cytoskeletal analysis: Immunofluorescence with phalloidin (for actin visualization) combined with CAF-1 antibody staining can reveal how CAF-1 levels affect cytoskeletal organization related to cell motility. Depletion of CAF-1 has been shown to disrupt typical stress fibers and promote podia-like actin structures associated with motile cells .

• Rescue experiments: Exogenous expression of CAF-1 subunits in cells where endogenous CAF-1 has been depleted can determine whether restoring CAF-1 levels reverses cancer-associated phenotypes, establishing causality rather than correlation.

How should researchers interpret discrepancies between CAF-1 antibody detection methods?

When faced with conflicting results from different detection methods:

• Western blot vs. immunofluorescence discrepancies: Western blotting detects the total protein amount, while immunofluorescence reveals subcellular localization. Differences may indicate epitope masking in certain cellular compartments or protein conformations. Consider using multiple antibodies targeting different epitopes of the same subunit to resolve these discrepancies.

• Antibody sensitivity differences: The SS48 antibody recognizes both full-length and truncated CAF-1, while other antibodies might be specific to certain forms or modified versions . Understanding each antibody's properties is essential for proper interpretation.

• Cell cycle considerations: CAF-1 activity is highest during S-phase. Results may vary depending on the cell cycle distribution of the population being studied. Synchronizing cells or performing cell cycle analysis alongside CAF-1 detection can clarify these variations.

What control experiments are essential when studying CAF-1 function using antibody-based methods?

Critical controls include:

• Antibody specificity controls: Include samples with siRNA-mediated depletion of the target CAF-1 subunit to confirm signal specificity.

• Expression rescue controls: When studying phenotypes after CAF-1 depletion, rescue experiments with siRNA-resistant CAF-1 constructs can confirm that observed effects are due to CAF-1 loss rather than off-target effects.

• Cell cycle controls: Since CAF-1 function is cell cycle-dependent, include analysis of cell cycle markers (e.g., PCNA for S-phase) to properly interpret results in the context of cell cycle position.

• Functional controls: In chromatin assembly assays, include samples with known assembly activity (positive control) and samples with CAF-1 immunodepleted (negative control) to validate the dynamic range of the assay .

How can researchers quantitatively analyze CAF-1 levels and activity in different experimental conditions?

Quantitative analysis approaches include:

• Western blot densitometry: Normalize CAF-1 subunit band intensities to stable loading controls (e.g., GAPDH or tubulin) across experimental conditions. This provides relative quantification of protein levels.

• Quantitative immunofluorescence: Measure mean fluorescence intensity of CAF-1 staining within defined cellular compartments (e.g., nucleus) across multiple cells. This approach can reveal subtle changes in protein levels or localization that might not be apparent in whole-cell lysate analysis.

• Activity assays quantification: For in vitro chromatin assembly assays, quantify the degree of supercoiling or the pattern of micrococcal nuclease digestion products to assess CAF-1 activity levels.

• Cell phenotype quantification: When studying CAF-1's role in processes like cell migration, quantitative measurements of wound closure rates or invasion assay results provide functional readouts that can be correlated with CAF-1 levels .