CBX5 Monoclonal Antibody

What is CBX5 and what are its key characteristics?

CBX5 (Chromobox Homolog 5) is a highly conserved nonhistone protein involved in heterochromatin formation and gene regulation. In humans, the canonical protein has a reported length of 191 amino acid residues and a molecular mass of 22.2 kDa, though it typically appears at 25-30 kDa in Western blots due to post-translational modifications . CBX5 is also known by several alternative names including HP1 alpha, HP1-ALPHA, HP1Hs alpha, antigen p25, and HEL25 .

CBX5 is primarily localized in the nucleus and is widely expressed across many tissue types . Functionally, it serves as a component of heterochromatin that recognizes and binds histone H3 tails methylated at 'Lys-9' (H3K9me), leading to epigenetic repression . The protein has been identified as a suppressor of metastasis, and its expression is down-regulated at both transcriptional and protein levels in metastatic compared to non-metastatic breast cancers .

What applications are most suitable for CBX5 monoclonal antibodies?

CBX5 monoclonal antibodies are versatile tools that can be used in multiple experimental applications. Based on extensive validation data, the following applications are well-established:

Western blotting is the most extensively validated application, with over 8 published studies confirming the utility of CBX5 antibodies in this context . Immunoprecipitation and ChIP applications have been validated in 2 and 1 published studies, respectively .

How should I validate the specificity of a CBX5 monoclonal antibody?

Thorough validation is essential to ensure the reliability of results obtained with CBX5 monoclonal antibodies. A comprehensive validation strategy should include:

• Positive control testing: Verify antibody performance in cell lines with known CBX5 expression. Validated cell lines include:

  • HeLa cells

  • Human kidney tissue

  • K-562 cells

  • A431 cells

  • MCF-7 cells

  • HEK-293 cells

  • U2OS cells

• Size verification: Confirm detection of a band at the expected molecular weight (25-30 kDa) in Western blot applications .

• Knockout/knockdown controls: Compare antibody signal in CBX5-expressing cells versus CBX5-depleted cells. At least one publication has validated CBX5 antibody specificity using knockdown/knockout approaches .

• Cross-reactivity assessment: Determine whether the antibody recognizes related proteins in the CBX family, which share structural similarities.

• Species reactivity confirmation: Verify reactivity with human, mouse, or rat samples as needed for your experimental system. Some antibodies show cross-reactivity across multiple species, while others are species-specific .

What are the optimal sample preparation protocols for CBX5 detection?

For robust and reproducible CBX5 detection, proper sample preparation is crucial:

For Western Blotting:

• Include protease inhibitors in lysis buffers to prevent degradation

• Ensure complete nuclear extraction, as CBX5 is primarily a nuclear protein

• Load 20-50 μg of total protein per lane

• Use freshly prepared samples when possible to maintain protein integrity

• For A431, U2OS, and HEK-293 cell lysates, a 1:1000 antibody dilution has been validated to detect CBX5 effectively

For Immunohistochemistry:

• Optimal antigen retrieval: TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

• Human lung cancer tissue has been validated as a positive control for IHC applications

• Dilution range of 1:50-1:500 is recommended, with optimization for specific tissues

For Immunofluorescence:

• Fixation with 4% paraformaldehyde preserves nuclear architecture for optimal CBX5 detection

• Nuclear counterstaining helps confirm the expected nuclear localization of CBX5

• A dilution of 1:100 has been validated for detecting CBX5 in U2OS cells

How can I detect post-translational modifications of CBX5?

CBX5 undergoes several post-translational modifications that affect its function and localization. To effectively study these modifications:

• Phosphorylation detection:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in lysis buffers

  • Consider phospho-specific antibodies if available for known CBX5 phosphorylation sites

  • The observed molecular weight shift from the calculated 22.2 kDa to the detected 25-30 kDa may partly result from phosphorylation events

• Ubiquitination detection:

  • Include deubiquitinase inhibitors in lysis buffers

  • Immunoprecipitate CBX5 under denaturing conditions before probing for ubiquitin

  • CBX5 has been reported to undergo ubiquitination, which can affect its stability and function

• Combined approaches:

  • Immunoprecipitate CBX5 before analyzing modifications to enrich the target protein

  • Use protein phosphatase treatments as controls to verify phosphorylation

  • Consider mass spectrometry approaches for comprehensive modification mapping

Understanding these modifications is crucial as they regulate CBX5's chromatin binding, protein interactions, and biological functions in different cellular contexts.

What strategies should I use for ChIP experiments investigating CBX5 binding to chromatin?

Chromatin Immunoprecipitation (ChIP) is valuable for studying CBX5's association with specific genomic regions. A methodological approach includes:

• Sample preparation optimization:

  • Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

  • Sonicate chromatin to 200-500 bp fragments for optimal immunoprecipitation

  • Include appropriate controls: input sample, IgG control, and positive control (e.g., H3K9me3 antibody)

• Immunoprecipitation considerations:

  • Use 0.5-4.0 μg of CBX5 monoclonal antibody per experiment, depending on antibody efficiency

  • Incubate overnight at 4°C for maximal binding

  • Use stringent washing to reduce background

• Analysis approaches:

  • For targeted analysis: qPCR of known heterochromatin regions or candidate target genes

  • For genome-wide analysis: ChIP-seq to map all CBX5 binding sites

  • Bioinformatic analysis should include correlation with repressive histone marks, particularly H3K9me3

CBX5 ChIP experiments have been successfully performed and published, validating the feasibility of this approach with specific antibodies .

How do I investigate the relationship between CBX5 expression and cancer progression?

CBX5 has significant implications in cancer biology, particularly as a suppressor of metastasis in breast cancer . To investigate this relationship:

• Expression analysis approaches:

  • IHC on tissue microarrays comparing primary tumors with metastatic lesions

  • Western blot analysis comparing cancer and normal tissues

  • Different cancer cell lines show varying levels of CBX5 expression that can be detected with monoclonal antibodies

• Prognostic correlation:

  • In breast cancer, CBX5 expression levels correlate with clinical outcomes in terms of patient survival

  • Expression also correlates with tumor size and disease stage

• Functional investigations:

  • Knockdown/knockout studies to assess CBX5's role in proliferation, migration, and invasion

  • Rescue experiments with wild-type CBX5 to confirm specificity of observed phenotypes

• Mechanistic studies:

  • ChIP-seq in cancer models to identify cancer-specific binding patterns

  • RNA-seq following CBX5 modulation to identify regulated genes

  • Co-immunoprecipitation to identify cancer-relevant protein interactions

The validated antibodies can detect CBX5 in various cancer cell lines and human cancer tissues, making them valuable tools for cancer research applications .

What are common issues with CBX5 antibody applications and how can I resolve them?

When working with CBX5 monoclonal antibodies, researchers may encounter several technical challenges:

• Multiple bands in Western blot:

  • Cause: Post-translational modifications, degradation, or non-specific binding

  • Solution: Include appropriate controls, optimize antibody dilution (1:500-1:1000), and ensure complete protease inhibition

• Weak signal in IHC/IF:

  • Cause: Insufficient antigen retrieval, low antibody concentration, or fixation issues

  • Solution: Optimize antigen retrieval using TE buffer pH 9.0 as recommended, adjust antibody concentration within the 1:50-1:500 range, and test different fixation protocols

• High background in immunostaining:

  • Cause: Non-specific binding, insufficient blocking, or excessive antibody

  • Solution: Increase blocking time/concentration, optimize antibody dilution, and include appropriate negative controls

• Inconsistent ChIP results:

  • Cause: Inefficient cross-linking, chromatin fragmentation issues, or antibody specificity

  • Solution: Optimize cross-linking conditions, verify chromatin fragmentation size, and validate antibody specificity in your experimental system

• Species cross-reactivity issues:

  • Cause: Antibody epitope specificity

  • Solution: Select antibodies validated for your species of interest. Some CBX5 antibodies show reactivity with human, mouse, and rat samples, while others are species-specific

How do I optimize CBX5 antibody dilution for my specific experimental system?

Optimization is essential for obtaining reliable results with CBX5 monoclonal antibodies:

• Western blot optimization:

  • Start with the recommended 1:1000 dilution that has been validated for multiple cell lines

  • Perform a dilution series (e.g., 1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio

  • Include positive control samples (HeLa, A431, or HEK-293 cell lysates)

  • Adjust exposure time to optimize signal detection

• IHC optimization:

  • Begin with a middle-range dilution (1:200) from the recommended 1:50-1:500 range

  • Test multiple antigen retrieval methods, with priority on TE buffer pH 9.0

  • Include positive control tissues (human lung cancer tissue has been validated)

  • Optimize incubation times and temperatures

• IF optimization:

  • Start with the validated 1:100 dilution for cell lines like U2OS

  • Test multiple fixation and permeabilization conditions

  • Optimize blocking to reduce background

  • Include appropriate negative controls

As noted in the antibody documentation, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

How can I use CBX5 antibodies to study epigenetic regulation mechanisms?

CBX5 monoclonal antibodies are powerful tools for investigating epigenetic regulatory mechanisms:

• Chromatin modification studies:

  • ChIP followed by qPCR or sequencing to map CBX5 binding sites genome-wide

  • Co-ChIP or sequential ChIP to investigate co-occupancy with other epigenetic factors

  • Correlation of CBX5 binding with repressive histone marks, particularly H3K9me3

• Protein interaction networks:

  • Immunoprecipitation with CBX5 antibodies to identify interacting proteins

  • Western blot analysis of co-immunoprecipitated factors

  • Mass spectrometry of immunoprecipitated complexes to discover novel interactions

• Functional genomics:

  • Combine CBX5 ChIP-seq with RNA-seq after CBX5 modulation to identify direct targets

  • Investigate CBX5 recruitment to specific genomic loci using ChIP-qPCR

  • Study changes in CBX5 localization under different cellular conditions

• Microscopy applications:

  • Immunofluorescence to study nuclear distribution patterns of CBX5

  • Co-localization with other heterochromatin markers

  • Live-cell imaging using CBX5 antibodies in conjunction with cell-permeable probes

These applications collectively provide insights into CBX5's role in establishing and maintaining heterochromatin domains and regulating gene expression.

What are the best approaches for studying CBX5 in cancer research models?

CBX5 has been identified as a suppressor of metastasis in breast cancer , making it an important target for cancer research:

• Expression analysis in cancer models:

  • Western blot analysis of CBX5 in cancer cell lines and tissues using 1:500-1:1000 antibody dilution

  • IHC staining of tumor tissues with 1:50-1:500 antibody dilution

  • Quantitative image analysis to correlate expression with clinical parameters

• Functional studies:

  • Knockdown/knockout followed by proliferation, migration, and invasion assays

  • Rescue experiments with wild-type or mutant CBX5

  • In vivo tumor models with CBX5 modulation

• Mechanistic investigations:

  • ChIP to identify cancer-specific binding patterns

  • Protein interaction studies in cancer versus normal contexts

  • Analysis of CBX5-regulated genes in cancer models

• Clinical correlations:

  • IHC on patient samples to correlate expression with outcomes

  • Analysis of CBX5 levels in relation to tumor size and stage

  • Investigation of CBX5 as a potential biomarker

Understanding CBX5's role in cancer progression may provide insights for diagnostic and therapeutic applications, particularly in breast cancer where its expression correlates with clinical outcomes .