CAF1-10 Antibody

What is CAF1-10 Antibody and what cellular processes does it target?

CAF1-10 Antibody is a research tool designed to detect components of the Chromatin Assembly Factor-1 (CAF-1) complex, which is essential for nucleosome assembly. The CAF-1 complex plays a crucial role in depositing newly synthesized and acetylated histones H3 and H4 into nascent chromatin during DNA replication. This process is vital for maintaining genomic integrity and regulating gene expression, as proper nucleosome assembly is essential for DNA accessibility to transcription factors and other regulatory proteins . The antibody specifically recognizes epitopes on CAF-1 components, enabling researchers to study chromatin assembly pathways, DNA replication processes, and epigenetic regulation mechanisms.

What are the different subunits of CAF-1 that can be detected with specific antibodies?

The CAF-1 complex consists of multiple subunits, each with distinct functions, that can be targeted by specific antibodies:

CAF-1 p60 antibodies detect this middle subunit in mouse, rat, and human samples through applications including western blotting, immunoprecipitation, immunofluorescence, and ELISA . Similarly, CAF-1 p150 antibodies specifically recognize the largest subunit of the complex, which has been validated in human and mouse samples .

What is the observed molecular weight of CAF-1 proteins in Western blot applications?

While calculated molecular weights provide theoretical values, the observed molecular weights in experimental settings may differ due to post-translational modifications or protein processing:

When troubleshooting unexpected band sizes, researchers should consider potential protein degradation, alternative splicing, post-translational modifications, or cross-reactivity with related proteins .

What are the validated applications for CAF-1 antibodies in research settings?

CAF-1 antibodies have been validated for multiple experimental applications with specific recommended protocols:

For Western blot applications, the CAF-1 p150 antibody has been successfully used with K562 cells , while CAF-1 p60 antibodies have been validated in mouse, rat, and human samples . These applications allow researchers to investigate CAF-1 expression, localization, and interaction partners in various experimental contexts.

How should I optimize immunoblot protocols for CAF-1 detection?

When optimizing immunoblot protocols for CAF-1 detection, researchers should consider the following methodological approach:

Sample preparation:

• Prepare total cell extracts by lysing cells in an appropriate buffer containing protease inhibitors

• For enhanced cross-linking studies, use dithiobis(succinimidyl propionate) at 2 mM for 15 minutes on ice, followed by quenching with 50 mM Tris pH 7.5

• Separate proteins by SDS-PAGE and transfer to nitrocellulose membranes

Antibody incubation parameters:

• For CAF-1 p150 detection: Use SS48 antibody at 1:1000 dilution, which recognizes both full-length and truncated forms

• For CAF-1 p60 detection: Use SS53 or a mixture of SS53 and SS96 monoclonal antibodies at 1:5000

• For general CAF-1 p150 detection using commercial antibodies: Start with 1:500-1:2000 dilution range and optimize based on signal intensity

Visualization systems:
Multiple conjugated antibody forms are available, including horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), phycoerythrin (PE), and Alexa Fluor conjugates, allowing flexibility in detection methodologies .

What are the critical considerations for CAF-1 antibody storage and handling?

Proper storage and handling are essential for maintaining antibody functionality and experimental reproducibility:

Additional recommendations:

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce binding efficacy

• Store antibodies in small aliquots to minimize freeze-thaw events

• CAF-1 p150 antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, which enhances stability

• When planning conjugation experiments, buffer exchange may be necessary to remove components like BSA or sodium azide that might interfere with conjugation chemistry

How can CAF-1 antibodies be used to study chromatin dynamics during DNA replication?

CAF-1 functions at the intersection of DNA replication and chromatin assembly, making it an excellent target for investigating replication-coupled chromatin dynamics:

Experimental approaches:

• Co-immunoprecipitation with CAF-1 antibodies to identify interaction partners during different stages of the cell cycle

• Chromatin immunoprecipitation (ChIP) to map CAF-1 occupancy on chromatin during DNA replication

• Proximity ligation assays to visualize CAF-1 association with replication forks in situ

The p60 subunit of CAF-1 is particularly involved in dynamic interactions with heterochromatin, which typically replicates later in S phase. By using CAF-1 p60 antibodies in synchronized cell populations, researchers can track the assembly of nucleosomes and heterochromatin proteins during DNA replication . This approach helps elucidate the mechanisms by which CAF-1 facilitates the orderly assembly of histones during replication, which is crucial for epigenetic inheritance and maintenance of genomic stability.

What are the implications of species cross-reactivity when using CAF-1 antibodies?

The sequence conservation of CAF-1 components across species affects antibody cross-reactivity and experimental design:

When considering using these antibodies in non-validated species:

• Perform sequence alignment (BLAST) between the immunogen sequence and the target species to assess potential cross-reactivity

• For CAF-1 p150 antibody, the immunogen corresponds to amino acids 300-380, so homology in this region is particularly important

• Conduct pilot experiments with positive controls from validated species alongside test samples

• Include appropriate negative controls to confirm specificity

As noted in the customer Q&A section, researchers have inquired about using CAF-1 p150 antibody in primate and dog tissues, suggesting interest in broader species applications despite limited validation data .

How can I investigate CAF-1 interactions with other chromatin-modifying complexes?

CAF-1 functionally interacts with multiple protein complexes involved in chromatin regulation, DNA repair, and epigenetic processes:

Methodological approach to study protein-protein interactions:

• Immunoprecipitation with anti-CAF-1 antibodies followed by mass spectrometry to identify novel interaction partners

• Cross-linking protocols can preserve transient interactions:

  • Trypsinize and wash cells in PBS

  • Resuspend in cold PBS and add dithiobis(succinimidyl propionate) to 2 mM

  • Incubate for 15 minutes on ice

  • Quench with Tris pH 7.5 to 50 mM

  • Prepare cell extracts and perform immunoprecipitation

Known interactions that can be studied include:

• PCNA complex - links CAF-1 to the replication fork

• KU complex components (KU70, KU80, DNA-PKCS) - implicated in DNA repair

• Inhibitor of histone acetyl transferases complex members (SET, ANP32A, ANP32B)

These interactions reveal CAF-1's involvement in coordinating nucleosome assembly with various nuclear processes, including DNA replication, repair, and epigenetic regulation.

What control experiments should be included when using CAF-1 antibodies?

Rigorous control experiments are essential for antibody validation and result interpretation:

Additionally:

• For polyclonal antibodies, include pre-immune serum controls where available

• In multiplexed experiments, include single-antibody controls to assess channel bleed-through

• When testing in non-validated species, include samples from validated species as reference points

These controls help distinguish specific signals from background and validate experimental findings, particularly when working with novel applications or sample types.

How can I troubleshoot unexpected band patterns in Western blot using CAF-1 antibodies?

Unexpected band patterns may result from various factors that should be systematically investigated:

For CAF-1 p150, the calculated molecular weight is 106.9 kDa, but the observed weight in Western blot may be around 39 kDa, which could represent a processed form or specific domain of the protein . When troubleshooting unexpected results, researchers should consider:

• Sample preparation methods that preserve protein integrity

• Using multiple antibodies targeting different epitopes of the same protein

• Validating results with complementary techniques (e.g., mass spectrometry, immunofluorescence)

• Consulting literature for reported molecular weights and band patterns

How are CAF-1 antibodies contributing to our understanding of epigenetic inheritance?

CAF-1 antibodies are instrumental in investigating the mechanisms of epigenetic inheritance during DNA replication:

Current research applications:

• Mapping the temporal and spatial dynamics of CAF-1 during cell cycle progression

• Investigating how CAF-1 coordinates with histone chaperones and chromatin remodelers

• Exploring CAF-1's role in maintaining heterochromatin states after replication

The p60 subunit of CAF-1 is particularly involved in dynamic interactions with heterochromatin, which is characterized by a tightly packed structure that generally replicates later in the S phase of the cell cycle. Using CAF-1 antibodies, researchers can track how CAF-1 facilitates the orderly assembly of histones and heterochromatin proteins during DNA replication, illuminating mechanisms of epigenetic regulation and cellular function .

What are the emerging techniques for studying CAF-1 dynamics in live cells?

While traditional antibody applications focus on fixed samples, emerging techniques are enabling dynamic studies of CAF-1 in living systems:

Future directions could involve:

• Development of cell-permeable antibodies or nanobodies for live-cell CAF-1 tracking

• Combining CAF-1 antibodies with super-resolution microscopy techniques to visualize assembly at the replication fork

• Utilizing split fluorescent protein systems to monitor CAF-1 subunit interactions in real time

• Developing FRET-based sensors to measure CAF-1 activity during chromatin assembly

These approaches would complement traditional antibody applications by providing dynamic information about CAF-1 behavior during cellular processes.