CHRAC1 Antibody

What is CHRAC1 and what cellular functions does it regulate?

CHRAC1 (also known as CHRAC15 or YCL1) is a 15 kDa subunit of the chromatin remodeling complex that plays essential roles in transcription and DNA replication . It functions as a component of ATP-dependent chromatin-remodeling complexes that regulate nucleosome spacing and accessibility. CHRAC1 is critical for:

• Chromatin structure maintenance

• Transcriptional regulation

• DNA replication processes

• Potentially DNA repair mechanisms

The protein consists of 131 amino acids with the sequence MADVVVGKDKGGEQRLISLPLSRIRVIMKSSPEVSSINQEALVLTAKATELFVQCLATYSYRHGSGKEKKVLTYSDLANTAQQSETFQFLADILPKKILASKYLKMLKEEKREEDEENDNDNESDHDEADS .

What is the cellular localization of CHRAC1 protein?

CHRAC1 is predominantly localized in the nucleus , consistent with its function in chromatin remodeling and transcriptional regulation. When detecting CHRAC1 via immunofluorescence, nuclear staining patterns should be expected. Understanding this localization is crucial for proper experimental design and interpretation of results.

What experimental applications are CHRAC1 antibodies suitable for?

Based on technical validation data, CHRAC1 antibodies have been successfully employed in:

Researchers should note that optimal dilutions may be sample-dependent and should be determined empirically for each application.

How should I validate the specificity of a CHRAC1 antibody?

A comprehensive validation approach should include:

• Knockout validation: Use CHRAC1 knockout/knockdown cells as negative controls. For example, using shRNA against CHRAC1 (e.g., shCHRAC1-1#: 5′-ACTCCACTGTCTCTAAGTAAA-3′) has been documented for specificity testing .

• Western blot analysis: Verify the antibody detects a single band at the expected molecular weight (~15 kDa) across multiple cell lines. Validated positive controls include HeLa, HEK-293, HepG2, K-562, and L02 cells .

• Immunoprecipitation followed by mass spectrometry: Confirm the antibody pulls down CHRAC1 and its known interaction partners.

• Signal reduction in knockdown experiments: Compare staining intensity between control and CHRAC1-depleted samples in your application of interest.

• Cross-reactivity assessment: Test the antibody across species if multi-species reactivity is claimed (human, mouse, rat) .

What are critical considerations when selecting a CHRAC1 antibody?

When selecting a CHRAC1 antibody, researchers should consider:

• Immunogen design: Antibodies raised against full-length protein or specific domains may have different epitope recognition properties. For example, some antibodies target amino acids 1-131 of human CHRAC1 (NP_059140.1) .

• Host species: Consider the host species (typically rabbit for polyclonal antibodies) in relation to your experimental design, especially for multi-color immunofluorescence.

• Polyclonal vs. monoclonal: Polyclonal antibodies offer broader epitope recognition but potentially more batch-to-batch variation compared to monoclonals.

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF, etc.).

• Species reactivity: Confirm reactivity with your experimental model organism (human, mouse, rat).

• Storage conditions: Most CHRAC1 antibodies require storage at -20°C in buffered solutions containing glycerol .

How can I optimize Western blot detection of CHRAC1?

For optimal Western blot detection of CHRAC1:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors and PMSF for cell lysis

  • Centrifuge at 12,000 rpm for 15 minutes at 4°C

• Electrophoresis conditions:

  • Use 10-15% SDS-PAGE gels (preferably 15% for better resolution of low molecular weight proteins)

  • Load 20-50 μg of total protein per lane

• Transfer parameters:

  • Transfer to PVDF membranes at 100V for 60-90 minutes

  • Verify transfer efficiency with Ponceau S staining

• Blocking and antibody incubation:

  • Block with 5-10% non-fat milk or BSA in TBST

  • Incubate with primary CHRAC1 antibody at 1:500-1:2000 dilution overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 dilution

• Detection:

  • Use ECL detection system for visualization

  • Expected band size is approximately 15 kDa

What are the best practices for studying CHRAC1 protein interactions?

To investigate CHRAC1 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Transfect cells with tagged constructs (e.g., Flag-CHRAC1 and HA-YAP)

  • Lyse cells in NP40 buffer

  • Incubate lysates with agarose beads and anti-Flag antibody or IgG control

  • Wash beads 4 times with NP40 buffer

  • Analyze by Western blot for interaction partners

• Proximity ligation assay (PLA):

  • Useful for detecting endogenous protein interactions in situ

  • Requires antibodies raised in different species

• Immunofluorescence co-localization:

  • Has been successfully used to show CHRAC1 co-localization with YAP

  • Use confocal microscopy for higher resolution

• Bio-ID or APEX proximity labeling:

  • These methods have identified CHRAC1 as a YAP interactor

  • Useful for identifying novel interaction partners

• GST pull-down assays:

  • For validating direct protein-protein interactions in vitro

How can I assess CHRAC1 function in chromatin remodeling?

To study CHRAC1's role in chromatin remodeling:

• ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing):

  • Compare chromatin accessibility profiles between control and CHRAC1-depleted cells

  • Analyze differential peaks to identify CHRAC1-dependent accessible regions

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing):

  • Use CHRAC1 antibodies to identify genomic binding sites

  • Compare with binding profiles of known interaction partners (e.g., YAP)

• DNase hypersensitivity assays:

  • Assess changes in chromatin accessibility following CHRAC1 manipulation

• Nucleosome positioning assays:

  • Analyze nucleosome occupancy and positioning in control versus CHRAC1-depleted cells

• Transcriptional reporter assays:

  • Measure the effect of CHRAC1 depletion on promoter activity of target genes

What is the role of CHRAC1 in cancer progression?

CHRAC1 has been implicated in cancer progression through several mechanisms:

How can I study CHRAC1-YAP interactions in cancer models?

To investigate the CHRAC1-YAP axis in cancer:

• Co-expression analysis:

  • Perform immunohistochemistry for both CHRAC1 and YAP in cancer tissues

  • Analyze correlation between expression patterns

  • Studies have shown significant correlation (p<0.0001) between YAP and CHRAC1 in breast and cervical cancer specimens

• Functional studies:

  • Use shRNA knockdown of CHRAC1 (e.g., shCHRAC1-1#: 5′-ACTCCACTGTCTCTAAGTAAA-3′)

  • Assess effects on:

    • YAP target gene expression via RT-qPCR

    • Cell proliferation using CCK-8 assays

    • Colony formation capacity

    • Tumor growth in xenograft models

• Mechanistic investigations:

  • Use Co-IP to confirm CHRAC1-YAP interaction

  • Perform immunofluorescence to visualize co-localization

  • Assess changes in YAP target gene expression following CHRAC1 manipulation

• Transcriptomic analysis:

  • Use RNA-seq to identify genes regulated by the CHRAC1-YAP axis

  • GSEA analysis to identify enriched pathways (e.g., Hippo pathway)

What are the key experimental models for studying CHRAC1 function?

Established experimental models for CHRAC1 research include:

• Cell line models:

  • Human breast cancer: MDA-MB-231

  • Human cervical cancer: HeLa

  • Human embryonic kidney: HEK-293T

  • Human liver cancer: HepG2

  • Human leukemia: K-562

• Gene manipulation approaches:

  • RNA interference: shRNA sequences targeting CHRAC1

  • CRISPR-Cas9 knockout: For complete ablation of CHRAC1 expression

• In vivo models:

  • Xenograft mouse models: BALB/c nude mice with CHRAC1-depleted cancer cells

  • Tumor formation assessed by volume (V = (L × W²) / 2) and weight

• Patient-derived samples:

  • Tissue microarrays of breast and cervical cancer specimens

  • Correlation studies with clinical parameters and survival data

What are common issues when detecting CHRAC1 and how can they be resolved?

How can I design rigorous CHRAC1 knockdown experiments?

For robust CHRAC1 knockdown studies:

• Use multiple shRNA/siRNA sequences:

  • Validated sequences: shCHRAC1-1#: 5′-ACTCCACTGTCTCTAAGTAAA-3′; shCHRAC1-2#: 5′-ACTCCACTGTCTCTAAGTAAA-3′

  • Include non-targeting control (shNC)

• Validation of knockdown efficiency:

  • Western blot to confirm protein reduction

  • RT-qPCR to verify mRNA depletion

  • Aim for >70% reduction in expression

• Rescue experiments:

  • Re-express shRNA-resistant CHRAC1 to confirm specificity

  • Should reverse phenotypic effects if they are specific to CHRAC1 loss

• Appropriate controls:

  • Use multiple cell lines to confirm biological relevance

  • Include time-course analysis for dynamic processes

• Functional readouts:

  • Proliferation assays (CCK-8, colony formation)

  • Gene expression analysis (RT-qPCR of target genes)

  • Phenotypic assays relevant to chromatin function

How should discrepancies in CHRAC1 research findings be interpreted?

When encountering contradictory findings in CHRAC1 research:

• Consider cell type specificity:

  • CHRAC1 function may vary between tissue types

  • Compare experimental conditions and cell models used

• Antibody differences:

  • Different antibodies may recognize distinct epitopes

  • Review immunogen sequences and validation data

  • Compare antibody performance across applications

• Experimental conditions:

  • Variations in knockdown efficiency

  • Differences in assay sensitivity and readouts

  • Timing of measurements (acute vs. chronic depletion)

• Data normalization approaches:

  • Review statistical methods used for analysis

  • Consider differences in reference genes or internal controls

• Contextual dependencies:

  • CHRAC1 may function differently depending on cellular context

  • Consider the influence of other pathway components or stress conditions

What are new methodologies for studying CHRAC1 chromatin functions?

Emerging techniques to investigate CHRAC1's role in chromatin dynamics include:

• CUT&RUN and CUT&Tag:

  • Higher signal-to-noise ratio than traditional ChIP-seq

  • Requires fewer cells and offers improved resolution

• Single-cell epigenomic approaches:

  • scATAC-seq to assess cell-to-cell variability in chromatin accessibility

  • Correlation with scRNA-seq for integrated analysis

• Hi-C and derivatives:

  • Study 3D chromatin organization changes upon CHRAC1 manipulation

  • Identify long-range chromatin interactions affected by CHRAC1

• Live-cell imaging of chromatin dynamics:

  • FRAP (Fluorescence Recovery After Photobleaching) to study CHRAC1 mobility

  • Fluorescently-tagged nucleosomes to track remodeling events

• Cryo-EM structural studies:

  • Resolve molecular structures of CHRAC1-containing complexes

  • Provide insights into interaction interfaces and functional mechanisms

How might CHRAC1 function as a therapeutic target in cancer?

Potential approaches to target CHRAC1 in cancer therapy include:

• Direct inhibition strategies:

  • Small molecule inhibitors of CHRAC1-YAP interaction

  • Peptide-based disruptors of protein-protein interactions

• Transcriptional regulation:

  • Epigenetic modulators to alter CHRAC1 expression

  • Promoter-targeted approaches (e.g., CRISPR interference)

• Combinatorial approaches:

  • Co-targeting CHRAC1 and YAP pathways

  • Combining with conventional chemotherapeutics

• Biomarker utilization:

  • CHRAC1 expression as predictor of therapy response

  • Patient stratification based on CHRAC1/YAP axis activation

• Delivery considerations:

  • Tumor-targeted delivery of CHRAC1 inhibitors

  • Cancer cell-specific expression systems