CAF1-11 Antibody

What is CAF1-11 and what is its functional role in plants?

CAF1-11 is a member of the CAF1 (CCR4-Associated Factor 1) protein family in plants. In Arabidopsis thaliana, CAF1 proteins function within the CCR4-NOT complex, which plays crucial roles in mRNA metabolism . While specific functions of the CAF1-11 isoform haven't been fully characterized, studies of CAF1 family members in Arabidopsis, rice, pepper, citrus, and poplar have demonstrated their involvement in both biotic and abiotic stress responses . These proteins primarily function in post-transcriptional regulation through mechanisms including mRNA deadenylation, which affects mRNA stability and decay.

What are recommended applications for CAF1-11 antibody in plant research?

The CAF1-11 antibody (product code: CSB-PA884302XA01DOA) is designed specifically for Arabidopsis thaliana research . Based on standard approaches for similar plant antibodies, recommended applications include:

• Western blotting to detect native CAF1-11 protein expression levels in plant tissues

• Immunoprecipitation to isolate CAF1-11 protein complexes

• Immunohistochemistry to visualize tissue localization patterns

• Chromatin immunoprecipitation (if CAF1-11 functions in chromatin-related processes)
  For optimal results, validation experiments should be performed in your specific experimental system before proceeding with full-scale studies.

How should I prepare plant samples for CAF1-11 antibody detection?

For optimal detection of CAF1-11 protein in Arabidopsis samples:

• Harvest fresh plant tissue and immediately freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining frozen state

• Extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect supernatant and quantify protein concentration

• For western blot applications, denature samples in SDS sample buffer
  Similar protein extraction methods have been successfully used for detecting related proteins in immunoblot studies .

What controls should be included when working with CAF1-11 antibody?

Including proper controls is essential for validating CAF1-11 antibody results:

• Positive control: Wild-type Arabidopsis thaliana tissue known to express CAF1-11

• Negative control:

  • Primary antibody omission

  • CAF1-11 knockdown/knockout tissue (if available)

  • Pre-immune serum control

• Loading control: Anti-actin or anti-tubulin antibody to verify equal protein loading

• Specificity control: Preincubation of antibody with recombinant CAF1-11 protein (peptide competition)
  These controls help distinguish specific from non-specific binding and validate experimental outcomes, particularly important given the existence of multiple CAF1 family members in Arabidopsis .

How can I distinguish between different CAF1 family members in Arabidopsis research?

Distinguishing between CAF1 family members is challenging due to possible sequence homology. A strategic approach includes:

• Sequence analysis: Perform alignment of CAF1 family sequences to identify unique epitopes for CAF1-11

• Western blot optimization:

  • Use gradient gels for better separation of similarly sized CAF1 proteins

  • Optimize transfer conditions for your protein size range

  • Titrate antibody concentrations to minimize cross-reactivity

• Validation through genetic approaches:

  • Use CAF1-11 overexpression lines as positive controls

  • Employ CAF1-11 knockout/knockdown lines as negative controls

  • Verify specificity through mass spectrometry analysis of immunoprecipitated proteins
    Research on TaCAF1 subfamily members has demonstrated that CAF1 proteins show more than 70% sequence identity within subfamilies , suggesting careful validation is needed for antibody specificity.

What approaches can be used to study CAF1-11's role in stress response pathways?

To investigate CAF1-11's role in plant stress responses:

• Expression analysis:

  • Monitor changes in CAF1-11 protein levels during stress exposure using the antibody

  • Compare with transcriptional changes using RT-qPCR

• Protein interaction studies:

  • Use CAF1-11 antibody for co-immunoprecipitation followed by mass spectrometry to identify stress-specific protein interactions

  • Verify interactions through techniques like BiFC or pull-down assays

• Functional studies:

  • Create CAF1-11 knockout/overexpression lines

  • Analyze phenotypes under different stress conditions

  • Perform genome-wide analysis of mRNA stability and decay rates

• Biochemical characterization:

  • Analyze deadenylation activity using in vitro assays with immunopurified complexes

  • Test nuclease activity on defined RNA substrates
    Studies in various plant species have demonstrated that CAF1 proteins play critical roles in both biotic and abiotic stress responses , making this a particularly valuable research direction.

How can I optimize immunoprecipitation protocols for CAF1-11 to identify novel interaction partners?

For effective immunoprecipitation (IP) of CAF1-11 and associated proteins:

• Crosslinking approach (for transient interactions):

  • Treat tissues with formaldehyde (1% for 10 minutes) or DSP (dithiobis-succinimidyl propionate)

  • Quench with glycine or Tris buffer

  • Extract proteins in RIPA buffer with protease inhibitors

• Native IP (for stable interactions):

  • Extract proteins in milder buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40)

  • Pre-clear lysate with protein A/G beads

  • Incubate with CAF1-11 antibody overnight at 4°C

• Analysis of interacting partners:

  • Elute complexes and analyze by mass spectrometry

  • Validate interactions by western blotting for suspected partners

  • Map interaction domains through deletion constructs
    This strategy is similar to approaches used for CAF1 complex characterization in other systems , which successfully identified interaction partners including components of DNA repair machinery and regulatory proteins.

What are the best approaches for studying CAF1-11's potential role in mRNA decay mechanisms?

To investigate CAF1-11's function in mRNA decay:

• RNA immunoprecipitation (RIP):

  • Use CAF1-11 antibody to pull down associated RNAs

  • Identify bound RNAs through sequencing (RIP-seq)

  • Validate specific targets using RT-qPCR

• Deadenylation assays:

  • Immunopurify CAF1-11 complexes using the antibody

  • Test deadenylation activity on radiolabeled substrates

  • Analyze reaction products by polyacrylamide gel electrophoresis

• In vivo mRNA stability assays:

  • Generate CAF1-11 knockdown/knockout lines

  • Measure half-lives of specific mRNAs following transcription inhibition

  • Compare poly(A) tail lengths using PAT assays or direct RNA sequencing

• Structure-function studies:

  • Analyze whether CAF1-11 contains conserved DEDD deadenylase domains

  • Create point mutations in catalytic residues

  • Test effects on deadenylation activity and mRNA stability
    Studies in other systems have shown that CAF1 depletion delayed deadenylation and degradation of constitutively expressed mRNAs and increased average poly(A) tail length , suggesting similar approaches would be valuable for CAF1-11 functional studies.

How can the CAF1-11 antibody be used in combination with other techniques to study chromatin dynamics?

If CAF1-11 functions in chromatin regulation (similar to CAF-1 in other systems), these approaches could be valuable:

• Chromatin immunoprecipitation (ChIP):

  • Use CAF1-11 antibody to immunoprecipitate protein-DNA complexes

  • Identify binding sites through sequencing (ChIP-seq)

  • Analyze enrichment at specific genomic features

• Proximity labeling:

  • Generate BioID or APEX2 fusions with CAF1-11

  • Identify proteins in close proximity during chromatin assembly

  • Validate interactions with CAF1-11 antibody

• ATAC-seq analysis in CAF1-11 mutants:

  • Generate CAF1-11 knockdown/knockout lines

  • Analyze changes in chromatin accessibility

  • Correlate with transcriptional changes

• Live-cell imaging:

  • Use CAF1-11 antibody for immunofluorescence studies

  • Analyze colocalization with DNA replication foci

  • Track dynamics during cell cycle progression
    Research has shown that CAF-1 suppression can trigger changes in chromatin accessibility at specific loci , suggesting CAF1-11 might have similar functions that could be investigated using these approaches.

What are the recommended troubleshooting steps for weak or non-specific CAF1-11 antibody signals?

When encountering issues with CAF1-11 antibody detection:

• For weak signals:

  • Increase antibody concentration (try 1:500 instead of 1:1000)

  • Extend incubation time (overnight at 4°C)

  • Use enhanced detection systems (e.g., SuperSignal West Femto)

  • Increase protein loading (50-100 μg per lane)

  • Try alternative extraction buffers to improve protein solubility

• For non-specific bands:

  • Increase blocking stringency (5% BSA or milk for 2 hours)

  • Add 0.1% Tween-20 to antibody dilution buffer

  • Optimize salt concentration in wash buffers (try 150-500 mM NaCl)

  • Use freshly prepared samples to prevent degradation

  • Try alternative blocking agents (normal serum from antibody host species)

• For high background:

  • Use shorter exposure times

  • Increase wash duration and number of washes

  • Filter antibody solution before use

  • Use fresh reagents and buffers
    Similar troubleshooting approaches have been effective for optimizing detection of other plant proteins in immunoblot studies .

What are effective strategies for validating CAF1-11 antibody specificity in Arabidopsis?

To validate the specificity of CAF1-11 antibody:

• Genetic validation:

  • Test antibody in CAF1-11 knockout/knockdown lines (expected: reduced/absent signal)

  • Test in CAF1-11 overexpression lines (expected: increased signal)

• Molecular validation:

  • Peptide competition assay using immunizing peptide

  • Western blot with recombinant CAF1-11 protein

  • Immunoprecipitation followed by mass spectrometry

• Cross-reactivity assessment:

  • Test against closely related CAF1 family members

  • Perform alignments to identify potential cross-reactive epitopes

  • Validate by testing antibody against recombinant versions of related proteins

• Orthogonal method validation:

  • Compare protein detection with mRNA expression by RT-qPCR

  • Tag CAF1-11 with epitope tags and compare detection patterns
    Researchers have used similar validation approaches for related proteins to ensure antibody specificity in complex biological samples .

How can CAF1-11 antibody be used to investigate evolutionary conservation of CAF1 function across plant species?

To explore evolutionary conservation of CAF1 function:

• Cross-species reactivity testing:

  • Test CAF1-11 antibody against protein extracts from diverse plant species

  • Analyze epitope conservation through sequence alignment

  • Optimize detection conditions for each species

• Comparative analysis:

  • Compare CAF1-11 expression patterns across species under various conditions

  • Analyze protein complexes across species through immunoprecipitation

  • Identify conserved vs. species-specific interactions

• Functional complementation:

  • Express Arabidopsis CAF1-11 in other species with CAF1 mutations

  • Use the antibody to confirm expression and localization

  • Assess functional rescue of phenotypes
    Genomic analysis has identified extensive duplication of CAF1 family members in wheat , suggesting evolutionary diversification that could be further explored using antibody-based approaches across species.

What experimental designs are most effective for studying CAF1-11's potential roles in DNA damage response?

If CAF1-11 functions similarly to CAF-1 in DNA repair processes, these approaches would be valuable:

• DNA damage induction experiments:

  • Treat plants with DNA-damaging agents (UV, bleomycin, MMS)

  • Monitor CAF1-11 localization using immunofluorescence

  • Analyze protein level changes by western blotting

• Chromatin association studies:

  • Perform chromatin fractionation before/after DNA damage

  • Use CAF1-11 antibody to detect recruitment to chromatin

  • Analyze co-recruitment with known DNA repair factors

• Protein interaction changes:

  • Immunoprecipitate CAF1-11 before/after DNA damage

  • Identify damage-specific interaction partners

  • Focus on potential interactions with DNA repair machinery

• Genetic interaction analysis:

  • Generate double mutants with DNA repair genes

  • Analyze synthetic phenotypes

  • Use the antibody to assess protein complex formation
    Studies have shown that CAF-1 is recruited to chromatin after DNA damage and can direct chromatin assembly on damaged DNA templates , suggesting similar approaches could reveal whether CAF1-11 has comparable functions.

How can phosphorylation state of CAF1-11 be investigated using the antibody?

To study CAF1-11 phosphorylation:

• Phosphorylation-state detection:

  • Treat protein extracts with/without phosphatase

  • Analyze mobility shifts by western blotting

  • Use Phos-tag gels for enhanced separation of phosphorylated forms

• Phosphorylation-specific interactome:

  • Immunoprecipitate CAF1-11 from tissues under different conditions

  • Test for interactions with phospho-binding proteins (e.g., 14-3-3 proteins)

  • Compare interactors before/after phosphatase treatment

• Kinase identification:

  • Perform in vitro kinase assays with immunopurified CAF1-11

  • Identify phosphorylation sites by mass spectrometry

  • Generate phospho-site mutants to test functional importance
    Research has shown that CAF-1 can be phosphorylated by DNA-dependent protein kinase, and this phosphorylation mediates interaction with 14-3-3 proteins , suggesting similar regulatory mechanisms might control CAF1-11 function.