CWF19L1 Antibody

What is CWF19L1 and why is it significant for research?

CWF19L1, also known as CWF19-like protein 1 or Cell Division Cycle Associated Protein 3, is a 538 amino acid protein belonging to the CWF19 family. It plays crucial roles in cell cycle regulation and RNA splicing, which are essential processes for maintaining cellular function and genomic stability . Recent research has identified CWF19L1 as a novel splicing factor that interacts with key spliceosomal proteins, including components of the U5 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA processing factor 19 (PRPF19) complex . The significance of CWF19L1 extends to immune regulation, where it enhances CD8+ T cell-mediated cytotoxicity against tumor cells, suggesting its potential as a therapeutic target in cancer immunotherapy .

What types of CWF19L1 antibodies are available for research applications?

Based on the available research tools, there are several types of CWF19L1 antibodies optimized for different applications:

How do I select the appropriate CWF19L1 antibody for my research?

When selecting a CWF19L1 antibody, consider the following factors:

• Experimental application: Different antibodies perform optimally in specific applications. For instance, while ab150841 is suitable for IHC-P and WB , the A-8 antibody offers broader application potential including IP and IF .

• Species reactivity: Ensure the antibody recognizes CWF19L1 in your experimental model. The available antibodies show reactivity to human samples, with some cross-reacting with mouse and rat samples .

• Epitope recognition: Consider which region of CWF19L1 the antibody targets. For studying specific domains or interactions, an antibody recognizing that particular region is preferable. For instance, ab150841 targets amino acids 100-250 .

• Validation status: Review available validation data, including Western blot images showing expected band sizes (approximately 61 kDa for CWF19L1) and IHC images demonstrating proper localization patterns.

How can CWF19L1 antibodies be utilized to study its role in T-cell cytotoxicity?

CWF19L1 has been recently identified as a promoter of T-cell mediated cytotoxicity against tumor cells. When investigating this function, researchers should consider:

• Flow cytometry analysis: Use CWF19L1 antibodies in conjunction with markers of T-cell activation to assess correlation between CWF19L1 expression and cytotoxic function.

• Co-immunoprecipitation studies: Employ CWF19L1 antibodies to pull down protein complexes and identify interaction partners within the cytotoxic pathway. Research has shown that CWF19L1 interacts with dozens of splicing factors and regulators, particularly components of U5 snRNP and the PRPF19 complex .

• Functional assays: Compare cytokine production in CWF19L1-expressing versus CWF19L1-deficient T cells. Studies have demonstrated that CWF19L1-deficient Jurkat cells show impaired production of effector cytokines including TNF-α, interferon-γ, and IL-2 upon activation .

• Tumor killing assays: CWF19L1-overexpressing cytotoxic T lymphocytes (CTLs) have shown enhanced killing specificity against multiple tumor cell lines compared to control CTLs . When designing such experiments, carefully control for antigen specificity and effector-to-target ratios.

What considerations are important when using CWF19L1 antibodies to study splicing regulation?

CWF19L1 functions as a novel splicing regulator, requiring specific experimental approaches:

• Nuclear/cytoplasmic fractionation: Given that CWF19L1 interacts with splicing factors in the nucleus, proper subcellular fractionation followed by immunoblotting is crucial to accurately assess its localization and interactions .

• RNA immunoprecipitation (RIP): Use CWF19L1 antibodies to precipitate RNA-protein complexes, followed by RNA sequencing to identify specific RNA targets regulated by CWF19L1.

• Alternative splicing analysis: When assessing CWF19L1's impact on alternative splicing, design primers spanning exon junctions and compare splicing patterns between wild-type and CWF19L1-deficient cells. Research has shown that CWF19L1 deficiency disrupts alternative splicing of immune-related genes .

• Splicing factor co-localization: Perform immunofluorescence studies using CWF19L1 antibodies alongside antibodies against known splicing factors (e.g., EFTUD2, SNRNP40, PRPF19, CDC5L, PQBP1) to assess their co-localization in nuclear speckles .

How should researchers approach investigating pathogenic CWF19L1 variants using antibodies?

CWF19L1 variants have been linked to autosomal recessive cerebellar ataxia (ARCA), presenting unique research challenges:

• Validation of variant expression: Use CWF19L1 antibodies to compare expression levels of wild-type versus mutant proteins. For instance, in cases with premature stop codons like the p.K316* variant , Western blotting may reveal truncated proteins or absence of expression.

• Functional domain analysis: Several pathogenic variants have been identified, including c.37G>C (p.D13H), c.946A>T (p.K316*) , c.1555_c.1557delGAG (exon 14), and c.1070G>T (exon 11) . Design experiments to assess how these mutations affect CWF19L1's interactions with splicing machinery.

• Tissue-specific expression: Compare CWF19L1 expression patterns in cerebellar tissue versus other tissues using immunohistochemistry to understand the neurological specificity of ARCA phenotypes.

• Protein stability assays: Employ pulse-chase experiments with immunoprecipitation using CWF19L1 antibodies to determine if pathogenic variants affect protein stability or turnover rates.

What are the optimal conditions for Western blotting with CWF19L1 antibodies?

For successful Western blot detection of CWF19L1:

• Sample preparation: Efficient lysis is crucial, especially for nuclear proteins like CWF19L1. Use RIPA buffer supplemented with protease inhibitors and DNase to ensure complete nuclear protein extraction.

• Running conditions: CWF19L1 has a predicted molecular weight of 61 kDa , making 10% SDS-PAGE gels appropriate for optimal resolution.

• Antibody dilutions: Different antibodies require specific working concentrations:

  • ab150841: 1/250 dilution for Western blotting

  • HPA036889: 0.04-0.4 μg/mL for immunoblotting

• Signal development: Both chemiluminescence and fluorescence-based detection systems are suitable, but fluorescence may offer advantages for quantitative analysis of expression levels.

• Positive controls: Include lysates from tissues known to express CWF19L1, such as human pancreas or specific cell lines like RT-4, U-251 MG, or NIH-3T3 cells that have demonstrated CWF19L1 expression .

How should immunohistochemistry experiments be designed when using CWF19L1 antibodies?

For optimal immunohistochemical detection of CWF19L1:

• Fixation and antigen retrieval: Formalin-fixed, paraffin-embedded (FFPE) samples typically require heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Test both to determine optimal conditions.

• Antibody dilutions:

  • ab150841: Use at 1/200 dilution for IHC-P

  • HPA036889: Use at 1:200-1:500 dilution for IHC

• Detection systems: Both DAB (3,3'-diaminobenzidine) and fluorescence-based systems work well, with the choice depending on whether co-localization studies are planned.

• Controls: Include positive control tissues such as human pancreas . Negative controls should omit primary antibody while maintaining all other steps.

• Counterstaining: For brightfield microscopy, hematoxylin counterstaining provides good nuclear context for assessing CWF19L1 expression patterns.

What are the key considerations for co-immunoprecipitation experiments with CWF19L1 antibodies?

When designing co-immunoprecipitation experiments to study CWF19L1 interactions:

• Lysis conditions: Use gentle lysis buffers (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) to preserve protein-protein interactions. Avoid harsh detergents that might disrupt the splicing complex interactions.

• Antibody selection: Choose antibodies validated for immunoprecipitation, such as the mouse monoclonal CWF19L1 Antibody (A-8) . Consider using antibodies targeting different epitopes to avoid interfering with specific protein interactions.

• Cross-linking: For transient or weak interactions, consider using chemical cross-linking agents like formaldehyde or DSP (dithiobis(succinimidyl propionate)) before cell lysis.

• Controls: Include IgG control immunoprecipitations and input samples. For studying specific interactions, such as with EFTUD2, SNRNP40, PRPF19, CDC5L, or PQBP1 , include reciprocal co-immunoprecipitations using antibodies against these proteins.

• Detection method: Western blotting following IP should use antibodies targeting different epitopes or species than those used for immunoprecipitation to avoid detecting the heavy and light chains of the IP antibody.

How can researchers verify the specificity of CWF19L1 antibodies?

Ensuring antibody specificity is critical for reliable results:

• Knockdown/knockout validation: Compare antibody signals in wild-type versus CWF19L1 knockdown/knockout samples. Complete signal loss in knockout samples strongly supports antibody specificity.

• Multiple antibody validation: Use multiple antibodies targeting different epitopes of CWF19L1 to confirm consistent patterns. Concordant results increase confidence in specificity.

• Mass spectrometry confirmation: Following immunoprecipitation with CWF19L1 antibodies, perform mass spectrometry analysis to confirm the identity of the precipitated protein.

• Blocking peptide competition: Pre-incubate the antibody with the immunogen peptide before application in your experiment. Specific signals should be significantly reduced or eliminated.

• Expected molecular weight verification: Confirm that the detected band appears at the expected molecular weight of 61 kDa for CWF19L1 . Multiple or unexpected bands may indicate cross-reactivity or protein degradation.

What are common pitfalls when working with CWF19L1 antibodies and how can they be addressed?

Several challenges may arise when working with CWF19L1 antibodies:

• Nuclear protein extraction efficiency: CWF19L1's nuclear localization may require optimization of nuclear extraction protocols. Insufficient extraction can lead to weak signals. Consider using specialized nuclear extraction kits or protocols.

• Cross-reactivity in immunohistochemistry: Background staining can occur, particularly in tissues with high endogenous peroxidase activity. Implement thorough blocking steps and optimize antibody concentration to improve signal-to-noise ratio.

• Epitope masking in co-immunoprecipitation: CWF19L1's interactions with splicing factors may mask antibody epitopes. Test multiple antibodies targeting different regions of the protein or use tagged constructs for pull-down experiments.

• Variability in expression levels: CWF19L1 expression may vary across tissues and cell types, requiring adjustment of antibody concentrations. Preliminary titration experiments can identify optimal working concentrations for each sample type.

• Antibody batch variation: Variation between antibody lots can affect results. Maintain detailed records of antibody batches used and consider bulk purchasing for long-term projects.

How can CWF19L1 antibodies contribute to understanding its role in cancer immunotherapy?

CWF19L1's emerging role in enhancing T-cell cytotoxicity opens several research avenues:

• Biomarker development: Investigate whether CWF19L1 expression levels correlate with immunotherapy response by analyzing tumor and immune cell samples from patients undergoing immunotherapy.

• Therapeutic target identification: Use CWF19L1 antibodies to identify and characterize downstream effectors of CWF19L1-mediated T-cell activation, potentially revealing new therapeutic targets.

• Tumor microenvironment analysis: Apply multiplex immunofluorescence with CWF19L1 antibodies to characterize its expression in tumor-infiltrating lymphocytes across different cancer types.

• Mechanistic studies: The finding that CWF19L1-overexpressing OT-I CTLs demonstrated significantly enhanced killing specificity against multiple tumor cell lines warrants further investigation into the mechanisms underlying this effect.

What experimental approaches could elucidate the connection between CWF19L1's splicing function and neurological disorders?

Given CWF19L1's association with autosomal recessive cerebellar ataxia:

• Isoform-specific detection: Develop antibodies or experimental approaches to distinguish between CWF19L1 splice variants in neuronal tissues to understand their differential roles.

• Neuronal model systems: Establish iPSC-derived neuronal models expressing wild-type versus pathogenic CWF19L1 variants to study functional consequences.

• Alternative splicing landscape: Use CWF19L1 antibodies in conjunction with RNA sequencing to compare global splicing patterns in normal versus pathogenic variant-expressing cells, focusing on genes involved in neuronal function.

• Protein-protein interaction networks: Compare CWF19L1 interactomes between neuronal and non-neuronal cells to identify tissue-specific interaction partners that might explain the neurological phenotype specificity.

• Therapeutic modulation: Explore whether modulation of CWF19L1 expression or function might restore proper splicing patterns in models of neurological disorders.